Chemical compositions and methods of using same

ABSTRACT

The present disclosure relates to chemical compositions, kits, and apparatuses and methods for using these compositions, kits and apparatuses in various assays.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. patent application Ser. No.15/819,151, filed Nov. 21, 2017 which claims priority to, and thebenefit of, U.S. Provisional Application No. 62/424,887, filed Nov. 21,2016; U.S. Provisional Application No. 62/457,237, filed Feb. 10, 2017;and U.S. Provisional Application No. 62/536,147, filed Jul. 24, 2017.The contents of each of the aforementioned patent applications areincorporated herein by reference in their entireties.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Sep. 3, 2019, isnamed NATE033-001US_SeqList_ST25.txt and is 19,394 bytes in size.

BACKGROUND OF THE INVENTION

There are currently a variety of methods for nucleic acid sequencing,i.e., the process of determining the precise order of nucleotides withina nucleic acid molecule. Current methods require amplifying a nucleicacid enzymatically, e.g., PCR, and/or by cloning. Further enzymaticpolymerizations are required to produce a detectable signal by a lightdetection means. Such amplification and polymerization steps are costlyand/or time-consuming. Thus, there is a need in the art for a method ofnucleic acid sequencing that is rapid and amplification- andenzyme-free. The present disclosure addresses these needs.

SUMMARY OF THE INVENTION

The present disclosure provides sequencing probes, methods, kits, andapparatuses that provide rapid enzyme-free, amplification-free, andlibrary-free nucleic acid sequencing that has long-read-lengths and withlow error rate. The sequencing probes described herein include barcodedomains in which each position in the barcode domain corresponds to atleast two nucleotides in the target binding domain. Moreover, themethods, kits, and apparatuses have rapid sample-to-answer capability.These features are particularly useful for sequencing in a clinicalsetting. The present disclosure is an improvement of the disclosuredisclosed in Patent Publication No. U.S. 2016/0194701, the contents ofwhich are herein incorporated by reference is their entirety.

The present disclosure provides a complex comprising a) a compositioncomprising a target binding domain and a barcode domain, wherein thetarget binding domain comprises at least eight nucleotides and iscapable of binding a target nucleic acid, wherein at least sixnucleotides in the target binding domain are capable of identifying acorresponding nucleotide in the target nucleic acid molecule and whereinat least two nucleotides in the target binding domain do not identify acorresponding nucleotide in the target nucleic acid molecule; wherein atleast two nucleotides of the at least six nucleotides in the targetbinding domain are modified nucleotides or nucleotide analogues; whereinthe barcode domain comprises a synthetic backbone, the barcode domaincomprising at least three attachment positions, each attachment positioncomprising at least one attachment region comprising at least onenucleic acid sequence capable of being bound by a complementary nucleicacid molecule, wherein the nucleic acid sequence of the at least threeattachment positions determines the position and identity of the atleast six nucleotides in the target nucleic acid that is bound by thetarget binding domain, and wherein each of the at least three attachmentpositions have a different nucleic acid sequence; and a firstcomplementary primary nucleic acid molecule hybridized to a firstattachment position of the at least three attachment positions, whereinthe first primary complementary nucleic acid molecule comprises at leasttwo domains and a linker modification, wherein the first domain ishybridized to the first attachment position of the barcode domain andthe second domain capable of hybridizing to at least one complementarysecondary nucleic acid molecule, and wherein the linker modification is

and wherein the linker modification is located between the first andsecond domains.

The present disclosure provides a complex comprising a) a compositioncomprising a target binding domain and a barcode domain, wherein thetarget binding domain comprises at least eight nucleotides and iscapable of binding a target nucleic acid, wherein at least sixnucleotides in the target binding domain are capable of identifying acorresponding nucleotide in the target nucleic acid molecule and whereinat least two nucleotides in the target binding domain do not identify acorresponding nucleotide in the target nucleic acid molecule; wherein atleast two nucleotides of the at least six nucleotides in the targetbinding domain are modified nucleotides or nucleotide analogues; whereinthe barcode domain comprises a synthetic backbone, the barcode domaincomprising at least three attachment positions, each attachment positioncomprising at least one attachment region comprising at least onenucleic acid sequence capable of being bound by a complementary nucleicacid molecule, wherein the nucleic acid sequence of the at least threeattachment positions determines the position and identity of the atleast six nucleotides in the target nucleic acid that is bound by thetarget binding domain, wherein each attachment position of the at leastthree attachment positions corresponds to two nucleotides of the atleast six nucleotides in the target binding domain and each of the atleast three attachment positions have a different nucleic acid sequence,and wherein the nucleic acid sequence of each position of the at leastthree attachment positions determines the position and identity of thecorresponding two nucleotides of the at least six nucleotides in thetarget nucleic acid that is bound by the target binding domain; and afirst complementary primary nucleic acid molecule hybridized to a firstattachment position of the at least three attachment positions, whereinthe first primary complementary nucleic acid molecule comprises at leasttwo domains and a linker modification, wherein the first domain ishybridized to the first attachment position of the barcode domain andthe second domain capable of hybridizing to at least one complementarysecondary nucleic acid molecule, and wherein the linker modification is

and wherein the linker modification is located between the first andsecond domains.

The present disclosure provides a sequencing probe comprising a targetbinding domain and a barcode domain; wherein the target binding domaincomprises at least eight nucleotides and is capable of binding a targetnucleic acid, wherein at least six nucleotides in the target bindingdomain are capable of identifying a corresponding nucleotide in thetarget nucleic acid molecule and wherein at least two nucleotides in thetarget binding domain do not identify a corresponding nucleotide in thetarget nucleic acid molecule; wherein at least two nucleotides of the atleast six nucleotides in the target binding domain are modifiednucleotides or nucleotide analogues; wherein the barcode domaincomprises a synthetic backbone, the barcode domain comprising at leastthree attachment positions, each attachment position comprising at leastone attachment region comprising at least one nucleic acid sequencecapable of being bound by a complementary nucleic acid molecule, whereinthe nucleic acid sequence of the at least three attachment positionsdetermines the position and identity of the at least six nucleotides inthe target nucleic acid that is bound by the target binding domain, andwherein each of the at least three attachment positions have a differentnucleic acid sequence.

The present disclosure provides a sequencing probe comprising a targetbinding domain and a barcode domain; wherein the target binding domaincomprises at least eight nucleotides and is capable of binding a targetnucleic acid, wherein at least six nucleotides in the target bindingdomain are capable of identifying a corresponding nucleotide in thetarget nucleic acid molecule and wherein at least two nucleotides in thetarget binding domain do not identify a corresponding nucleotide in thetarget nucleic acid molecule; wherein at least two nucleotides of the atleast six nucleotides in the target binding domain are modifiednucleotides or nucleotide analogues; wherein the barcode domaincomprises a synthetic backbone, the barcode domain comprising at leastthree attachment positions, each attachment position comprising at leastone attachment region comprising at least one nucleic acid sequencecapable of being bound by a complementary nucleic acid molecule, whereineach attachment position of the at least three attachment positionscorresponds to two nucleotides of the at least six nucleotides in thetarget binding domain and each of the at least three attachmentpositions have a different nucleic acid sequence, and wherein thenucleic acid sequence of each position of the at least three attachmentpositions determines the position and identity of the corresponding twonucleotides of the at least six nucleotides in the target nucleic acidthat is bound by the target binding domain.

The present disclosure provides a sequencing probe comprising a targetbinding domain and a barcode domain; wherein the target binding domaincomprises at least ten nucleotides and is capable of binding a targetnucleic acid, wherein at least six nucleotides in the target bindingdomain are capable of identifying a corresponding nucleotide in thetarget nucleic acid molecule and wherein at least four nucleotides inthe target binding domain do not identify a corresponding nucleotide inthe target nucleic acid molecule; wherein the barcode domain comprises asynthetic backbone, the barcode domain comprising at least threeattachment positions, each attachment position comprising at least oneattachment region comprising at least one nucleic acid sequence capableof being bound by a complementary nucleic acid molecule, wherein thenucleic acid sequence of the at least three attachment positionsdetermines the position and identity of the at least six nucleotides inthe target nucleic acid that is bound by the target binding domain, andwherein each of the at least three attachment positions have a differentnucleic acid sequence.

The present disclosure also provides a sequencing probe comprising atarget binding domain and a barcode domain; wherein the target bindingdomain comprises at least ten nucleotides and is capable of binding atarget nucleic acid, wherein at least six nucleotides in the targetbinding domain are capable of identifying a corresponding nucleotide inthe target nucleic acid molecule and wherein at least four nucleotidesin the target binding domain do not identify a corresponding nucleotidein the target nucleic acid molecule; wherein the barcode domaincomprises a synthetic backbone, the barcode domain comprising at leastthree attachment positions, each attachment position comprising at leastone attachment region comprising at least one nucleic acid sequencecapable of being bound by a complementary nucleic acid molecule, whereineach attachment position of the at least three attachment positionscorresponds to two nucleotides of the at least six nucleotides in thetarget binding domain and each of the at least three attachmentpositions have a different nucleic acid sequence, and wherein thenucleic acid sequence of each position of the at least three attachmentpositions determines the position and identity of the corresponding twonucleotides of the at least six nucleotides in the target nucleic acidthat is bound by the target binding domain.

The synthetic backbone can comprise a polysaccharide, a polynucleotide,a peptide, a peptide nucleic acid, or a polypeptide. The syntheticbackbone can comprise DNA. The synthetic backbone comprises asingle-stranded DNA. The sequencing probe can comprise a single-strandedDNA synthetic backbone and a double-stranded DNA spacer between thetarget binding domain and the barcode domain. The double-stranded DNAspacer can be from about 1 nucleotide to about 100 nucleotides inlength; from about 2 nucleotides to about 50 nucleotides in length; orfrom 20 nucleotides to 40 nucleotides in length. The double-stranded DNAspacer is about 36 nucleotides in length. The sequencing probe cancomprise a single-stranded DNA synthetic backbone and a polymer-basedspacer between the target binding domain and the barcode domain, whereinthe polymer-based spacer provides similar mechanical properties as adouble-stranded DNA spacer.

The number of nucleotides in the target binding domain can be the sameas, less than, or greater than, the number of attachment positions inthe barcode domain. Preferably, the number of nucleotides in the targetbinding domain is greater than the number of attachment positions in thebarcode domain. The number of nucleotides in the target binding domaincan be at least three more, at least four more, at least five more, atleast six more, at least seven more, at least eight more, at least ninemore or at least ten more than the number of attachment positions in thebarcode domain. Preferably, the target binding domain comprises eightnucleotides and the barcode domain comprises three attachment positions.

At least three, at least four, at least five, or at least sixnucleotides in the target binding domain capable of identifying acorresponding nucleotide in the target nucleic acid molecule can bemodified nucleotides or nucleotide analogues. The modified nucleotide ornucleic acid analogue can be locked nucleic acids (LNA), bridged nucleicacids (BNA), propyne-modified nucleic acids, zip nucleic acids (ZNA®),isoguanine, isocytosine, or any combination thereof. Preferably, themodified nucleotide or nucleic acid analogue is a locked nucleic acid(LNA).

The at least two nucleotides in the target binding domain that do notidentify a corresponding nucleotide can precede the at least sixnucleotides in the target binding domain. The at least two nucleotidesin the target binding domain that do not identify a correspondingnucleotide can follow the at least six nucleotides in the target bindingdomain. The at least one of the at least two nucleotides in the targetbinding domain that do not identify a corresponding nucleotide precedesthe at least six nucleotides in the target binding domain and wherein atleast one of the at least two nucleotides in the target binding domainthat do not identify a corresponding nucleotide follows the at least sixnucleotides in the target binding domain. That is, the at least twonucleotides in the target binding domain that do not identify acorresponding nucleotide flank the at least six nucleotides in thetarget binding domain.

The at least two nucleotides in the target binding domain that do notidentify a corresponding nucleotide in the target nucleic acid moleculecan be universal bases, degenerate bases, or a combination thereof. Atleast two of the at least four nucleotides in the target binding domainthat do not identify a corresponding nucleotide in the target nucleicacid molecule can be universal bases, degenerate bases, or a combinationthereof.

Each attachment position in the barcode domain can comprise oneattachment region. Each attachment position in the barcode domain cancomprise more than one attachment region.

Each attachment position in the barcode domain can comprise the samenumber of attachment regions. Each attachment position in the barcodedomain can comprise a different number of attachment regions. At leastone of the at least three attachment positions in the barcode domain cancomprise a different number of attachment regions than the other two.When the attachment position in the barcode domain comprises more thanone attachment region, the attachment regions can be the same. When theattachment position in the barcode domain comprises more than oneattachment region, the attachment regions can comprise the same nucleicacid sequence. When the attachment position in the barcode domaincomprises more than one attachment region, the attachment regions can bedifferent. When the attachment position in the barcode domain comprisesmore than one attachment region, the attachment regions can comprise adifferent nucleic acid sequence.

Each nucleic acid sequence comprising each attachment region in thebarcode domain is from about 8 nucleotides to about 20 nucleotides inlength. Each nucleic acid sequence comprising each attachment region inthe barcode domain is about 12 nucleotides in length. Each nucleic acidsequence comprising each attachment region in the barcode domain isabout 14 nucleotides in length.

At least one, at least two, or at least three of the at least threeattachment positions in the barcode domain can be adjacent to at leastone flanking single-stranded polynucleotide. Each of the at least threeattachment positions in the barcode domain can be adjacent to at leastone flanking single stranded polynucleotide.

At least one, at least two, or at least three attachment regions in atleast one attachment position can be integral to the synthetic backbone.Each attachment region in each of the at least three attachmentpositions can be integral to the synthetic backbone. At least one, atleast two, or at least three attachment regions in at least oneattachment position can branch from the synthetic backbone. Eachattachment region in each of the at least three attachment positions canbranch from the synthetic backbone.

The complementary nucleic acid molecule capable of binding, directly orindirectly, to at least one nucleic acid sequence within at least oneattachment region within each attachment position can be RNA, DNA orPNA. Preferably, the complementary nucleic acid molecule is DNA.

The complementary nucleic acid molecule can be a primary nucleic acidmolecule, wherein a primary nucleic acid molecule directly binds to atleast one attachment region within at least one attachment position ofthe barcode domain.

The primary nucleic acid molecule can comprise at least two domains, afirst domain capable of binding to at least one attachment region withinat least one attachment position of the barcode domain and a seconddomain capable of binding to at least one complementary secondarynucleic acid molecule. The primary nucleic acid molecule can comprise atleast two domains, a first domain capable of binding to at least oneattachment region within at least one attachment position of the barcodedomain and a second domain comprising comprises a first detectable labeland an at least second detectable label.

The primary nucleic molecule can be hybridized to at least oneattachment region within at least one attachment position of the barcodedomain and be hybridized to at least one, at least two, at least three,at least four, at least five, or more secondary nucleic acid molecules.Preferably the primary nucleic molecule is hybridized to four secondarynucleic acid molecules. The primary nucleic molecule can be hybridizedto at least one attachment region within at least one attachmentposition of the barcode domain and can be hybridized to a firstdetectable label and an at least second detectable label. The first andat least second detectable labels can have the same emission spectrum orcan have different emission spectra

The primary nucleic acid molecule can comprise a cleavable linker.Preferably, the cleavable linker is located between the first domain andthe second domain. The cleavable linker can be a photo-cleavable linker(e.g., UV-light cleavable linker), a reducing agent cleavable linker, oran enzymatically cleavable linker. Preferably, the linker is aphoto-cleavable linker.

A secondary nucleic acid molecule can comprise at least two domains, afirst domain capable of binding to complementary sequence in at leastone primary nucleic acid molecule; and a second domain capable ofbinding to (a) a first detectable label and an at least seconddetectable label, (b) to at least one complementary tertiary nucleicacid molecule, or (c) a combination thereof.

The secondary nucleic molecule can be hybridized to at least one primarynucleic acid molecule and be hybridized to at least one, at least two,at least three, at least four, at least five, at least six, at leastseven or more tertiary nucleic acid molecules. Preferably the secondarynucleic molecule is hybridized to one tertiary nucleic acid molecule.The secondary nucleic molecule can be hybridized to at least one primarynucleic acid molecule and can comprise a first detectable label and anat least second detectable label. The secondary nucleic molecule can behybridized to at least one primary nucleic acid molecule, at least onetertiary nucleic acid molecule and to a first detectable label and an atleast second detectable label. The first and at least second detectablelabels can have the same emission spectrum or can have differentemission spectra. When the secondary nucleic molecule is hybridized toat least one primary nucleic acid molecule, at least one tertiarynucleic acid molecule comprising a first detectable label and an atleast second detectable label, and to a first detectable label and an atleast second detectable label, the at least first and second detectablelabels located on the secondary nucleic acid molecule can have the sameemission spectra and the at least first and second detectable labelslocated on the tertiary nucleic acid molecule can have the same emissionspectra, and wherein the emission spectra of the detectable labels onthe secondary nucleic acid molecule can be different than the emissionspectra of the detectable labels on the tertiary nucleic acid molecule.

The secondary nucleic acid molecule can comprise a cleavable linker.Preferably, the cleavable linker is located between the first domain andthe second domain. The cleavable linker can be a photo-cleavable linker(e.g., UV-light cleavable linker), a reducing agent cleavable linker, oran enzymatically cleavable linker. Preferably, the linker is aphoto-cleavable linker.

A tertiary nucleic acid molecule can comprise at least two domains, afirst domain capable of binding to complementary sequence in at leastone secondary nucleic acid molecule; and a second domain capable ofbinding to a first detectable label and an at least second detectablelabel.

The tertiary nucleic molecule can be hybridized to at least onesecondary nucleic acid molecule and can comprise a first detectablelabel and an at least second detectable label. The first and at leastsecond detectable labels can have the same emission spectrum or can havedifferent emission spectra.

The tertiary nucleic acid molecule can comprise a cleavable linker.Preferably, the cleavable linker is located between the first domain andthe second domain. The cleavable linker can be a photo-cleavable linker(e.g., UV-light cleavable linker), a reducing agent cleavable linker, oran enzymatically cleavable linker. Preferably, the linker is aphoto-cleavable linker.

The present disclosure also provides a population of the sequencingprobes comprising a plurality of the sequencing probes disclosed herein.Preferably, each sequencing probe within the plurality of sequencingprobes comprises a different target binding domain and binds a differentregion within a target nucleic acid.

The present disclosure also provides a method for sequencing a nucleicacid comprising (1) hybridizing a sequencing probe described herein to atarget nucleic acid that is optionally immobilized to a substrate at oneor more positions; (2) binding a first complementary nucleic acidmolecule comprising a first detectable label and an at least seconddetectable label to a first attachment position of the at least threeattachment positions of the barcode domain; (3) detecting the first andat least second detectable label of the bound first complementarynucleic acid molecule; (4) identifying the position and identity of atleast two nucleotides in the immobilized target nucleic acid; (5)binding to the first attachment position a first hybridizing nucleicacid molecule lacking a detectable label, thereby unbinding the firstcomplementary nucleic acid molecule comprising the detectable labels, orcontacting the first complementary nucleic acid molecule comprising thedetectable labels with a force sufficient to release the firstdetectable label and at least second detectable label; (6) binding asecond complementary nucleic acid molecule comprising a third detectablelabel and an at least fourth detectable label to a second attachmentposition of the at least three attachment positions of the barcodedomain; (7) detecting the third and at least fourth detectable label ofthe bound second complementary nucleic acid molecule; (8) identifyingthe position and identity of at least two nucleotides in the optionallyimmobilized target nucleic acid; (9) repeating steps (5) to (8) untileach attachment position of the at least three attachment positions inthe barcode domain have been bound by a complementary nucleic acidmolecule comprising two detectable labels, and the two detectable labelsof the bound complementary nucleic acid molecule has been detected,thereby identifying the linear order of at least six nucleotides for atleast a first region of the optionally immobilized target nucleic acidthat was hybridized to the target binding domain of the sequencingprobe; and (10) removing the sequencing probe from the optionallyimmobilized target nucleic acid.

The method can further comprise (11) hybridizing a second sequencingprobe to a target nucleic acid that is optionally immobilized to asubstrate at one or more positions, and wherein the target bindingdomain of the first sequencing probe and the second sequencing probe aredifferent; (12) binding a first complementary nucleic acid moleculecomprising a first detectable label and an at least second detectablelabel to a first attachment position of the at least three attachmentpositions of the barcode domain; (13) detecting the first and at leastsecond detectable label of the bound first complementary nucleic acidmolecule; (14) identifying the position and identity of at least twonucleotides in the optionally immobilized target nucleic acid; (15)binding to the first attachment position a first hybridizing nucleicacid molecule lacking a detectable label, thereby unbinding the firstcomplementary nucleic acid molecule or complex comprising the detectablelabels, or contacting the first complementary nucleic acid molecule orcomplex comprising the detectable labels with a force sufficient torelease the first detectable label and at least second detectable label;(16) binding a second complementary nucleic acid molecule comprising athird detectable label and an at least fourth detectable label to asecond attachment position of the at least three attachment positions ofthe barcode domain; (17) detecting the third and at least fourthdetectable label of the bound second complementary nucleic acidmolecule; (18) identifying the position and identity of at least twonucleotides in the optionally immobilized target nucleic acid; (19)repeating steps (15) to (18) until each attachment position of the atleast three attachment positions in the barcode domain have been boundby a complementary nucleic acid molecule comprising two detectablelabels, and the two detectable labels of the bound complementary nucleicacid molecule has been detected, thereby identifying the linear order ofat least six nucleotides for at least a second region of the optionallyimmobilized target nucleic acid that was hybridized by the targetbinding domain of the sequencing probe; and (20) removing the secondsequencing probe from the optionally immobilized target nucleic acid.

The method can further comprise assembling each identified linear orderof nucleotides in the at least first region and at least second regionof the immobilized target nucleic acid, thereby identifying a sequencefor the immobilized target nucleic acid.

Steps (5) and (6) can occur sequentially or concurrently. The first andat least second detectable labels can have the same emission spectrum orcan have different emission spectra. The third and at least fourthdetectable labels can have the same emission spectrum or can havedifferent emission spectra.

The first complementary nucleic acid molecule can comprise a cleavablelinker. The second complementary nucleic acid molecule can comprise acleavable linker. The first complementary nucleic acid molecule and thesecond complementary nucleic acid molecule can each comprise a cleavablelinker. Preferably, the cleavable linker is photo-cleavable. The releaseforce can be light. Preferably, UV light. The light can be provided by alight source selected from the group consisting of an arc-lamp, a laser,a focused UV light source, and light emitting diode.

The first complementary nucleic acid molecule and the first hybridizingnucleic acid molecule lacking a detectable label can comprise the samenucleic acid sequence. The first hybridizing nucleic acid moleculelacking a detectable label can comprise a nucleic acid sequencecomplementary to a flanking single-stranded polynucleotide adjacent tothe first attachment position in the barcode domain.

The second complementary nucleic acid molecule and the secondhybridizing nucleic acid molecule lacking a detectable label cancomprise the same nucleic acid sequence. The second hybridizing nucleicacid molecule lacking a detectable label can comprise a nucleic acidsequence complementary to a flanking single-stranded polynucleotideadjacent to the second attachment position in the barcode domain.

The present disclosure also provides a method for sequencing a nucleicacid comprising (1) hybridizing at least one first population of firstsequencing probes comprising a plurality of the sequencing probesdescribed herein to a target nucleic acid that is optionally immobilizedto a substrate at one or more positions; (2) binding a firstcomplementary nucleic acid molecule comprising a first detectable labeland an at least second detectable label to a first attachment positionof the at least three attachment positions of the barcode domain; (3)detecting the first and at least second detectable label of the boundfirst complementary nucleic acid molecule; (4) identifying the positionand identity of at least two nucleotides in the optionally immobilizedtarget nucleic acid; (5) binding to the first attachment position afirst hybridizing nucleic acid molecule lacking a detectable label,thereby unbinding the first complementary nucleic acid moleculecomprising the detectable labels, or contacting the first complementarynucleic acid molecule comprising the detectable labels with a forcesufficient to release the first detectable label and at least seconddetectable label; (6) binding a second complementary nucleic acidmolecule comprising a third detectable label and an at least fourthdetectable to a second attachment position of the at least threeattachment positions of the barcode domain; (7) detecting the third andat least fourth detectable label of the bound second complementarynucleic acid molecule; (8) identifying the position and identity of atleast two nucleotides in the optionally immobilized target nucleic acid;(9) repeating steps (5) to (8) until each attachment position of the atleast three attachment positions in the barcode domain have been boundby a complementary nucleic acid molecule comprising two detectablelabels, and the two detectable labels of the bound complementary nucleicacid molecule has been detected, thereby identifying the linear order ofat least six nucleotides for at least a first region of the optionallyimmobilized target nucleic acid that was hybridized by the targetbinding domain of the sequencing probe; and (10) removing the at leastone first population of first sequencing probes from the optionallyimmobilized target nucleic acid.

The method can further comprise (11) hybridizing at least one secondpopulation of second sequencing probes comprising a plurality of thesequencing probes disclosed herein to a target nucleic acid that isoptionally immobilized to a substrate at one or more positions, andwherein the target binding domain of the first sequencing probe and thesecond sequencing probe are different; (12) binding a firstcomplementary nucleic acid molecule comprising a first detectable labeland an at least second detectable label to a first attachment positionof the at least three attachment positions of the barcode domain; (13)detecting the first and at least second detectable label of the boundfirst complementary nucleic acid molecule; (14) identifying the positionand identity of at least two nucleotides in the optionally immobilizedtarget nucleic acid; (15) binding to the first attachment position afirst hybridizing nucleic acid molecule lacking a detectable label,thereby unbinding the first complementary nucleic acid molecule orcomplex comprising the detectable labels, or contacting the firstcomplementary nucleic acid molecule or complex comprising the detectablelabels with a force sufficient to release the first detectable label andat least second detectable label; (16) binding a second complementarynucleic acid molecule comprising a third detectable label and an atleast fourth detectable label to a second attachment position of the atleast three attachment positions of the barcode domain; (17) detectingthe third and at least fourth detectable label of the bound secondcomplementary nucleic acid molecule; (18) identifying the position andidentity of at least two nucleotides in the optionally immobilizedtarget nucleic acid; (19) repeating steps (15) to (18) until eachattachment position of the at least three attachment positions in thebarcode domain have been bound by a complementary nucleic acid moleculecomprising two detectable labels, and the two detectable labels of thebound complementary nucleic acid molecule has been detected, therebyidentifying the linear order of at least six nucleotides for at least asecond region of the optionally immobilized target nucleic acid that washybridized by the target binding domain of the sequencing probe; and(20) removing the at least one second population of second sequencingprobes from the optionally immobilized target nucleic acid.

The method can further comprise assembling each identified linear orderof nucleotides in the at least first region and at least second regionof the immobilized target nucleic acid, thereby identifying a sequencefor the immobilized target nucleic acid.

Steps (5) and (6) can occur sequentially or concurrently. The first andat least second detectable labels can have the same emission spectrum orcan have different emission spectra. The third and at least fourthdetectable labels can have the same emission spectrum or can havedifferent emission spectra.

The first complementary nucleic acid molecule can comprise a cleavablelinker. The second complementary nucleic acid molecule can comprise acleavable linker. The first complementary nucleic acid molecule and thesecond complementary nucleic acid molecule can each comprise a cleavablelinker. Preferably, the cleavable linker is photo-cleavable. The releaseforce can be light. Preferably, UV light. The light can be provided by alight source selected from the group consisting of an arc-lamp, a laser,a focused UV light source, and light emitting diode.

The first complementary nucleic acid molecule and the first hybridizingnucleic acid molecule lacking a detectable label can comprise the samenucleic acid sequence. The first hybridizing nucleic acid moleculelacking a detectable label can comprise a nucleic acid sequencecomplementary to a flanking single-stranded polynucleotide adjacent tothe first attachment position in the barcode domain.

The second complementary nucleic acid molecule and the secondhybridizing nucleic acid molecule lacking a detectable label cancomprise the same nucleic acid sequence. The second hybridizing nucleicacid molecule lacking a detectable label can comprise a nucleic acidsequence complementary to a flanking single-stranded polynucleotideadjacent to the second attachment position in the barcode domain.

The present disclosure also provides a method for determining anucleotide sequence of a nucleic acid comprising (1) hybridizing a firstsequencing probe as disclosed herein to a target nucleic acid that isoptionally immobilized to a substrate at one or more positions; (2)hybridizing a first complementary nucleic acid molecule comprising afirst detectable label and a second detectable label to a firstattachment position of the at least three attachment positions of thebarcode domain; (3) identifying the first and the second detectablelabel of the first complementary nucleic acid molecule hybridized to thefirst attachment position; (4) removing the first and the seconddetectable label hybridized to the first attachment position; (5)hybridizing a second complementary nucleic acid molecule comprising athird detectable label and a fourth detectable label to a secondattachment position of the at least three attachment positions of thebarcode domain; (6) identifying the third and the fourth detectablelabel of the second complementary nucleic acid molecule hybridized tothe second attachment position; (7) removing the third and fourthdetectable label hybridized to the second attachment position; (8)hybridizing a third complementary nucleic acid molecule comprising afifth detectable label and a sixth detectable label to a thirdattachment position of the at least three attachment positions of thebarcode domain; (9) identifying the fifth and the sixth detectable labelof the third complementary nucleic acid molecule hybridized to the thirdattachment position; and (10) identifying the linear order of at leastsix nucleotides of the optionally immobilized target nucleic acidhybridized to the target binding domain of the sequencing probe based onthe identity of the first detectable label, second detectable label,third detectable label, fourth detectable label, fifth detectable labeland sixth detectable label.

The method can further comprise (11) removing the at least firstsequencing probe from the first region of the optionally immobilizedtarget nucleic acid; (12) hybridizing at least a second sequencing probeas disclosed herein to a second region of the target nucleic acid thatis optionally immobilized to a substrate at one or more positions, andwherein the target binding domain of the first sequencing probe and theat least second sequencing probe are different; —(13) hybridizing afirst complementary nucleic acid molecule comprising a first detectablelabel and a second detectable label to a first attachment position ofthe at least three attachment positions of the barcode domain; (14)detecting the first and the second detectable label of the firstcomplementary nucleic acid molecule hybridized to the first attachmentposition; (15) hybridizing a second complementary nucleic acid moleculecomprising a third detectable label and a fourth detectable label to asecond attachment position of the at least three attachment positions ofthe barcode domain; (16) detecting the third and the fourth detectablelabel of the second complementary nucleic acid molecule hybridized tothe second attachment position; (17) removing the third and fourthdetectable label hybridized to the second attachment position; (18)hybridizing a third complementary nucleic acid molecule comprising afifth detectable label and a sixth detectable label to a thirdattachment position of the at least three attachment positions of thebarcode domain; (19) identifying the fifth and the sixth detectablelabel of the third complementary nucleic acid molecule hybridized to thethird attachment position; and (20) identifying the linear order of atleast six nucleotides in the second region of the optionally immobilizedtarget nucleic acid hybridized to the target binding domain of the atleast second sequencing probe based on the identity of the firstdetectable label, second detectable label, third detectable label,fourth detectable label, fifth detectable label and sixth detectablelabel.

The method can further comprise assembling each identified linear orderof nucleotides in the at least first region and at least second regionof the immobilized target nucleic acid, thereby identifying a sequencefor the immobilized target nucleic acid.

Steps (4) and (5) can occur sequentially or concurrently. The first andat least second detectable labels can have the same emission spectrum orcan have different emission spectra. The third and at least fourthdetectable labels can have the same emission spectrum or can havedifferent emission spectra. The fifth and at least sixth detectablelabels can have the same emission spectrum or can have differentemission spectra.

The first complementary nucleic acid molecule can comprise a cleavablelinker. The second complementary nucleic acid molecule can comprise acleavable linker. The third complementary nucleic acid molecule cancomprise a cleavable linker. The first complementary nucleic acidmolecule, the second complementary nucleic acid molecule and the atleast third complementary nucleic molecule can each comprise a cleavablelinker. Preferably, the cleavable linker is photo-cleavable. A method ofremoving any one of the first complementary nucleic acid molecule, thesecond complementary nucleic acid molecule and the at least thirdcomplementary nucleic molecule can comprise contact with light.Preferably, UV light. The light can be provided by a light sourceselected from the group consisting of an arc-lamp, a laser, a focused UVlight source, and light emitting diode.

The present disclosure also provides a method for determining thenucleotide sequence of a nucleic acid comprising (1) hybridizing a firstsequencing probe as disclosed herein to a first region of a targetnucleic acid obtained from a predetermine gene, wherein the targetnucleic acid is optionally immobilized to a substrate at one or morepositions; (2) hybridizing a first complementary nucleic acid moleculecomprising a first detectable label and a second detectable label to afirst attachment position of the at least three attachment positions ofthe barcode domain; (3) detecting the first and the second detectablelabel of the first complementary nucleic acid molecule hybridized to thefirst attachment position; (4) removing the first and the seconddetectable label hybridized to the first attachment position; (5)hybridizing a second complementary nucleic acid molecule comprising athird detectable label and a fourth detectable label to a secondattachment position of the at least three attachment positions of thebarcode domain; (6) detecting the third and and fourth detectable labelof the second complementary nucleic acid molecule hybridized to thesecond attachment position; (7) removing the third and the fourthdetectable label hybridized to the second attachment position; (8)hybridizing a third complementary nucleic acid molecule comprising afifth detectable label and a sixth detectable label to a thirdattachment position of the at least three attachment positions of thebarcode domain; (9) identifying the fifth and the sixth detectable labelof the third complementary nucleic acid molecule hybridized to the thirdattachment position; and (10) identifying the linear order of the atleast six nucleotides in the first region of the optionally immobilizedtarget nucleic acid hybridized to the target binding domain of the firstsequencing probe based on the identity of the first detectable label,second detectable label, third detectable label, fourth detectablelabel, fifth detectable label and sixth detectable label.

The method can further comprise (11) removing the at least firstsequencing probe from the first region of the optionally immobilizedtarget nucleic acid; (12) hybridizing at least a second sequencing probeas disclosed herein to a second region of the target nucleic acid thatis optionally immobilized to a substrate at one or more positions, andwherein the target binding domain of the first sequencing probe and theat least second sequencing probe are different; —(13) hybridizing afirst complementary nucleic acid molecule comprising a first detectablelabel and a second detectable label to a first attachment position ofthe at least three attachment positions of the barcode domain; (14)detecting the first and the second detectable label of the firstcomplementary nucleic acid molecule hybridized to the first attachmentposition; (15) hybridizing a second complementary nucleic acid moleculecomprising a third detectable label and a fourth detectable label to asecond attachment position of the at least three attachment positions ofthe barcode domain; (16) detecting the third and the fourth detectablelabel of the second complementary nucleic acid molecule hybridized tothe second attachment position; (17) removing the third and fourthdetectable label hybridized to the second attachment position; (18)hybridizing a third complementary nucleic acid molecule comprising afifth detectable label and a sixth detectable label to a thirdattachment position of the at least three attachment positions of thebarcode domain; (19) identifying the fifth and the sixth detectablelabel of the third complementary nucleic acid molecule hybridized to thethird attachment position; and (20) identifying the linear order of atleast six nucleotides in the second region of the optionally immobilizedtarget nucleic acid hybridized to the target binding domain of the atleast second sequencing probe based on the identity of the firstdetectable label, second detectable label, third detectable label,fourth detectable label, fifth detectable label and sixth detectablelabel.

The method can further comprise assembling each identified linear orderof nucleotides in the at least first region and at least second regionof the immobilized target nucleic acid, thereby identifying a sequencefor the immobilized target nucleic acid.

Steps (4) and (5) can occur sequentially or concurrently. The first andat least second detectable labels can have the same emission spectrum orcan have different emission spectra. The third and at least fourthdetectable labels can have the same emission spectrum or can havedifferent emission spectra. The fifth and at least sixth detectablelabels can have the same emission spectrum or can have differentemission spectra.

The first complementary nucleic acid molecule can comprise a cleavablelinker. The second complementary nucleic acid molecule can comprise acleavable linker. The third complementary nucleic acid molecule cancomprise a cleavable linker. The first complementary nucleic acidmolecule, the second complementary nucleic acid molecule and the atleast third complementary nucleic molecule can each comprise a cleavablelinker. Preferably, the cleavable linker is photo-cleavable. A method ofremoving any one of the first complementary nucleic acid molecule, thesecond complementary nucleic acid molecule and the at least thirdcomplementary nucleic molecule can comprise contact with light.Preferably, UV light. The light can be provided by a light sourceselected from the group consisting of an arc-lamp, a laser, a focused UVlight source, and light emitting diode.

The present disclosure also provides an apparatus for performing any ofthe methods disclosed herein.

The present disclosure also provides one or more kits comprising asubstrate, a population of sequencing probes disclosed herein, at leastthree complementary nucleic acid molecules comprising a first detectablelabel and at least two second detectable labels, and instructions foruse. The one or more kits can further comprise at least one captureprobe. The one or more kits can further comprise at least two captureprobes.

Any of the above aspects can be combined with any other aspect.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs. In the Specification, thesingular forms also include the plural unless the context clearlydictates otherwise; as examples, the terms “a,” “an,” and “the” areunderstood to be singular or plural and the term “or” is understood tobe inclusive. By way of example, “an element” means one or more element.Throughout the specification the word “comprising,” or variations suchas “comprises” or “comprising,” will be understood to imply theinclusion of a stated element, integer or step, or group of elements,integers or steps, but not the exclusion of any other element, integeror step, or group of elements, integers or steps. About can beunderstood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%,0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear fromthe context, all numerical values provided herein are modified by theterm “about.”

Although methods and materials similar or equivalent to those describedherein can be used in the practice or testing of the present disclosure,suitable methods and materials are described below. All publications,patent applications, patents, and other references mentioned herein areincorporated by reference in their entirety. The references cited hereinare not admitted to be prior art to the claimed invention. In the caseof conflict, the present Specification, including definitions, willcontrol. In addition, the materials, methods, and examples areillustrative only and are not intended to be limiting. Other featuresand advantages of the disclosure will be apparent from the followingdetailed description and claim.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawings will be provided by the Office upon request and paymentof the necessary fee.

The above and further features will be more clearly appreciated from thefollowing detailed description when taken in conjunction with theaccompanying drawings.

FIG. 1 is an illustration of one exemplary sequencing probe of thepresent disclosure.

FIG. 2 shows the design of standard and three-part sequencing probes ofthe present disclosure.

FIG. 3 is an illustration of an exemplary reporter complex of thepresent disclosure hybridized to an exemplary sequencing probe of thepresent disclosure.

FIG. 4 shows a schematic illustration of an exemplary reporter probe ofthe present disclosure.

FIG. 5 is a schematic illustration of several exemplary reporter probesof the present disclosure.

FIG. 6 is a schematic illustration of exemplary reporter probes of thepresent disclosure comprising “extra-handles”.

FIG. 7 is a schematic illustration of several exemplary reporter probesof the present disclosure comprising different arrangements of tertiarynucleic acids.

FIG. 8 is a schematic illustration of several exemplary reporter probesof the present disclosure comprising branching tertiary nucleic acids.

FIG. 9 is an illustration of one exemplary reporter probe of the presentdisclosure comprising a cleavable linker modification.

FIG. 10 shows possible positions for cleavable linker modificationswithin an exemplary reporter probe of the present disclosure.

FIG. 11 is a schematic illustration of the capture of a target nucleicacid using the two capture probe system of the present disclosure.

FIG. 12 shows the results from an experiment using the present methodsto capture and detect a multiplex cancer panel, composed of 100 targets,using a FFPE sample.

FIG. 13 shows a schematic illustration of two captured target DNAmolecules hybridized to capture probes, blocker oligos, and sequencingprobes for targeted sequencing of large target nucleic acid molecules.

FIG. 14 is a schematic illustration of a single cycle of the sequencingmethod of the present disclosure.

FIG. 15 is a schematic illustration of one cycle of the sequencingmethod of the present disclosure and the corresponding imaging datacollected during this cycle.

FIG. 16 illustrates an exemplary sequencing probe pool configuration ofthe present disclosure in which the eight color combinations are used todesign eight different pools of sequencing probes.

FIG. 17 compares the barcode domain design disclosed in U.S. 2016/019470with the barcode domain design of the present disclosure.

FIG. 18 is a schematic illustration of a single sequencing probe or aplurality of sequencing probes of the present disclosure hybridized to acaptured target nucleic acid molecule.

FIG. 19 shows fluorescence images recorded during the sequencing methodof the present disclosure when a single sequencing probe or a pluralityof sequencing probes are hybridized to a target nucleic acid.

FIG. 20 is a schematic illustration of a plurality of sequencing probesof the present disclosure bound along the length of a target nucleicacid and the corresponding recorded fluorescence images.

FIG. 21 shows exemplary imaging data recorded during a sequencing cycleof the present disclosure and the fluorescence signal intensity profilesof reporter probes of the present disclosure.

FIG. 22 is a schematic illustration of a sequencing cycle of the presentdisclosure in which a cleavable linker modification is used to darken abarcode position.

FIG. 23 is an illustrative example of an exemplary sequencing cycle ofthe present disclosure in which a position within a barcode domain isdarkened by displacement of the primary nucleic acids.

FIG. 24 shows an example of integrated capture of RNA and DNA from aFFPE sample.

FIG. 25 is schematic illustration of how the sequencing method of thepresent disclosure allows for the sequencing of the same base of atarget nucleic acid with different sequencing probes.

FIG. 26 shows how multiple base calls for a specific nucleotide positionon the target nucleic acid, recorded from one or more sequencing probes,can be combined to create a consensus sequence, thereby increasing theaccuracy of the final base call.

FIG. 27 shows recorded fluorescence images of the sequencing method ofthe present disclosure after the capture, stretching, and detection of33 kilobase DNA fragments.

FIG. 28 shows the results from a sequencing experiment obtained usingthe sequencing method of the present disclosure and analyzed using theShortStack™ algorithm. For plots on the left panel, starting at the topleft plot proceeding clockwise, sequences shown correspond to SEQ IDNOs: 3, 4, 6, 8, 7 and 5. For the table on the right, starting at thetop moving down, sequences correspond to SEQ ID NOs: 3, 4, 7, 8, 6 and5.

FIG. 29 shows a schematic illustration of the experimental design forthe multiplexed capture and sequencing of oncogene targets from a FFPEsample.

FIG. 30 shows an illustrative schematic of direct RNA sequencing and theresults from experiments to test the compatibility of RNA molecules withthe sequencing method of the present disclosure.

FIG. 31 shows the sequencing of a RNA molecule and a DNA molecule thathave the same nucleotide sequence using the sequencing method of thepresent disclosure.

FIG. 32 shows the results of a multiplex target capture of an RNA panel.

FIG. 33 shows a schematic overview of the steps of the ShortStack™software pipeline.

FIG. 34 shows the results of mutational analysis of simulated data setsusing the ShortStack™ software pipeline.

FIG. 35 shows the overall mutation calling accuracy of the ShortStack™software pipeline for various types of mutations.

FIG. 36 the intensity distributions for a reporter complex labeled withparticular color combinations.

FIG. 37 shows a typical deposition gradient of the present disclosure.

FIG. 38 shows the capture efficiency of the present disclosure during atitration of DNA mass inputs between 25 ng to 500 ng.

FIG. 39 shows the HLPC purification of an exemplary reporter complex ofthe present disclosure.

FIG. 40 shows hybridization efficiencies and accuracy of reporter probesof the present disclosure in the presence of different buffer additives.

FIG. 41 shows percentage of target nucleic acid loss when the sequencingmethod of the present disclosure is used in the presence of differentbuffer additives.

FIG. 42 shows efficiency and error of exemplary reporter probes of thepresent disclosure that comprise a 12mer complementary nucleic acid.

FIG. 43 shows efficiency and error of reporter probes using an exemplary8×8×8 14mer reporter set.

FIG. 44 shows efficiency and error of reporter probes using an exemplary10×10×10 14mer reporter set.

FIG. 45 shows a comparison of the performance of standard and three-partsequencing probes of the present disclosure.

FIG. 46 shows the effect of LNA substitutions within exemplary targetbinding domains of the present disclosure using individual probes.

FIG. 47 shows the effect of LNA substitutions within exemplary targetbinding domains of the present disclosure using a pool of nine probes.

FIG. 48 shows the effect of modified nucleotides and nucleic acidanalogue substitutions in exemplary target binding domains of thepresent disclosure.

FIG. 49 shows the results from an experiment to quantify the rawaccuracy of the sequencing method of the present disclosure

FIG. 50 shows the results from an experiment to determine the accuracyof the sequencing method of the present disclosure when nucleotides inthe target nucleic acid are sequenced by more than one sequencing probe.

DETAILED DESCRIPTION OF THE INVENTION

The present disclosure provides sequencing probes, reporter probes,methods, kits, and apparatuses that provide rapid, enzyme-free,amplification-free, and library-free nucleic acid sequencing that haslong-read-lengths and with low error rate.

Compositions of the Present Disclosure

The present disclosure provides a sequencing probe comprising a targetbinding domain and a barcode domain; wherein the target binding domaincomprises at least eight nucleotides and is capable of binding a targetnucleic acid, wherein at least six nucleotides in the target bindingdomain are capable of identifying a corresponding (complementary)nucleotide in the target nucleic acid molecule and wherein at least twonucleotides in the target binding domain do not identify a correspondingnucleotide in the target nucleic acid molecule; wherein at least one, orat least two nucleotides, of the at least six nucleotides in the targetbinding domain are modified nucleotides or nucleotide analogues andwherein the at least one, or at least two nucleotides in the targetbinding domain are universal or degenerate bases; wherein the barcodedomain comprises a synthetic backbone, the barcode domain comprising atleast three attachment positions, each attachment position comprising atleast one attachment region comprising at least one nucleic acidsequence capable of being bound by a complementary nucleic acidmolecule, wherein each attachment position of the at least threeattachment positions corresponds to two nucleotides of the at least sixnucleotides in the target binding domain and each of the at least threeattachment positions have a different nucleic acid sequence, and whereinthe nucleic acid sequence of each position of the at least threeattachment positions determines the position and identity of thecorresponding two nucleotides of the at least six nucleotides in thetarget nucleic acid that is bound by the target binding domain.

The present disclosure also provides a sequencing probe comprising atarget binding domain and a barcode domain; wherein the target bindingdomain comprises at least ten nucleotides and is capable of binding atarget nucleic acid, wherein at least six nucleotides in the targetbinding domain are capable of identifying a corresponding(complementary) nucleotide in the target nucleic acid molecule andwherein at least four nucleotides in the target binding domain do notidentify a corresponding nucleotide in the target nucleic acid molecule;wherein the barcode domain comprises a synthetic backbone, the barcodedomain comprising at least three attachment positions, each attachmentposition comprising at least one attachment region comprising at leastone nucleic acid sequence capable of being bound by a complementarynucleic acid molecule, wherein each attachment position of the at leastthree attachment positions corresponds to two nucleotides of the atleast six nucleotides in the target binding domain and each of the atleast three attachment positions have a different nucleic acid sequence,and wherein the nucleic acid sequence of each position of the at leastthree attachment positions determines the position and identity of thecorresponding two nucleotides of the at least six nucleotides in thetarget nucleic acid that is bound by the target binding domain.

The present disclosure also provides a population of sequencing probescomprising a plurality of any of the sequencing probes disclosed herein.

The target binding domain, barcode domain, and backbone of the disclosedsequencing probes, as well as, the complementary nucleic acid molecule(e.g., reporter molecules or reporter complexes) are described in moredetail below.

A sequencing probe of the present disclosure comprises a target bindingdomain and a barcode domain. FIG. 1 is a schematic illustration of anexemplary sequencing probe of the present disclosure. FIG. 1 shows thatthe target binding domain is capable of binding a target nucleic acid. Atarget nucleic acid can be any nucleic acid to which the sequencingprobe of the present disclosure can hybridize. The target nucleic acidcan be DNA or RNA. The target nucleic acid can be obtained from abiological sample from a subject. The terms “target binding domain” and“sequencing domain” are used interchangeably herein.

The target binding domain can comprise a series of nucleotides (e.g. isa polynucleotide). The target binding domain can comprise DNA, RNA, or acombination thereof. In the case when the target binding domain is apolynucleotide, the target binding domain binds to a target nucleic acidby hybridizing to a portion of the target nucleic acid that iscomplementary to the target binding domain of the sequencing probe, asshown in FIG. 1.

The target binding domain of the sequencing probe can be designed tocontrol the likelihood of sequencing probe hybridization and/orde-hybridization and the rates at which these occur. Generally, thelower a probe's Tm, the faster and more likely that the probe willde-hybridize to/from a target nucleic acid. Thus, use of lower Tm probeswill decrease the number of probes bound to a target nucleic acid.

The length of a target binding domain, in part, affects the likelihoodof a probe hybridizing and remaining hybridized to a target nucleicacid. Generally, the longer (greater number of nucleotides) a targetbinding domain is, the less likely that a complementary sequence will bepresent in the target nucleotide. Conversely, the shorter a targetbinding domain is, the more likely that a complementary sequence will bepresent in the target nucleotide. For example, there is a 1/256 chancethat a four-mer sequence will be located in a target nucleic acid versusa 1/4096 chance that a six-mer sequence will be located in the targetnucleic acid. Consequently, a collection of shorter probes will likelybind in more locations for a given stretch of a nucleic acid whencompared to a collection of longer probes.

In circumstances, it is preferable to have probes having shorter targetbinding domains to increase the number of reads in the given stretch ofthe nucleic acid, thereby enriching coverage of a target nucleic acid ora portion of the target nucleic acid, especially a portion of particularinterest, e.g., when detecting a mutation or SNP allele.

The target binding domain can be any amount or number of nucleotides inlength. The target binding domain can be at least 12 nucleotides inlength, at least 10 nucleotides in length, at least 8 nucleotides inlength, at least 6 nucleotides in length or at least three nucleotidesin length.

Each nucleotide in the target binding domain can identify (or code for)a complementary nucleotide of the target molecule. Alternatively, somenucleotides in the target binding domain identify (or code for) acomplementary nucleotide of the target molecule and some nucleotides inthe target binding domain do not identify (or code for) a complementarynucleotide of the target molecule.

The target binding domain can comprise at least one natural base. Thetarget binding domain can comprise no natural bases. The target bindingdomain can comprise at least one modified nucleotide or nucleic acidanalog. The target binding domain can comprise no modified nucleotidesor nucleic acid analogs. The target binding domain can comprise at leastone universal base. The target binding domain can comprise no universalbases. The target binding domain can comprise at least one degeneratebase. The target binding domain can comprise no degenerate bases.

The target domain can comprise any combination natural bases (e.g. 0, 1,2, 3, 4, 5, 6, 7, 8, 9, 10, or more natural bases), modified nucleotidesor nucleic acid analogs (e.g. 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or moremodified or analog nucleotides), universal bases (e.g. 0, 1, 2, 3, 4, 5,6, 7, 8, 9, 10, or more universal bases), or degenerate bases (e.g. 0,1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more degenerative bases). When presentin a combination, the natural bases, modified nucleotides or nucleicacid analogs, universal bases and degenerate bases of a particulartarget binding domain can be arranged in any order.

The terms “modified nucleotides” or “nucleic acid analogues” include,but are not limited to, locked nucleic acids (LNA), bridged nucleicacids (BNA), propyne-modified nucleic acids, zip nucleic acids (ZNA®),isoguanine and isocytosine. The target binding domain can include zeroto six (e.g. 0, 1, 2, 3, 4, 5 or 6) modified nucleotides or nucleic acidanalogues. Preferably, the modified nucleotides or nucleic acidanalogues are locked nucleic acids (LNAs).

The term “locked nucleic acids (LNA)” as used herein includes, but isnot limited to, a modified RNA nucleotide in which the ribose moietycomprises a methylene bridge connecting the 2′ oxygen and the 4′ carbon.This methylene bridge locks the ribose in the 3′-endo confirmation, alsoknown as the north confirmation, that is found in A-form RNA duplexes.The term inaccessible RNA can be used interchangeably with LNA. The term“bridged nucleic acids (BNA)” as used herein includes, but is notlimited to, modified RNA molecules that comprise a five-membered orsix-membered bridged structure with a fixed 3′-endo confirmation, alsoknown as the north confirmation. The bridged structure connects the 2′oxygen of the ribose to the 4′ carbon of the ribose. Various differentbridge structures are possible containing carbon, nitrogen, and hydrogenatoms. The term “propyne-modified nucleic acids” as used hereinincludes, but is not limited to, pyrimidines, namely cytosine andthymine/uracil, that comprise a propyne modification at the C5 positionof the nucleic acid base. The term “zip nucleic acids (ZNA®)” as usedherein includes, but is not limited to, oligonucleotides that areconjugated with cationic spermine moieties.

The term “universal base” as used herein includes, but is not limitedto, a nucleotide base does not follow Watson-Crick base pair rules butrather can bind to any of the four canonical bases (A, T/U, C, G)located on the target nucleic acid. The term “degenerate base” as usedherein includes, but is not limited to, a nucleotide base that does notfollow Watson-Crick base pair rules but rather can bind to at least twoof the four canonical bases A, T/U, C, G), but not all four. Adegenerate base can also be termed a Wobble base; these terms are usedinterchangeably herein.

The exemplary sequencing probe depicted in FIG. 1 illustrates a targetbinding domain that comprises a six nucleotide long (6-mer) sequence(b₁-b₂-b₃-b₆) that hybridizes specifically to complementary nucleotides1-6 of the target nucleic acid that is to be sequenced. This 6-merportion of the target binding domain (b₁-b₂-b₃-b₆) identifies (or codesfor) the complementary nucleotides in the target sequence (1-2-3-4-5-6).This 6-mer sequence is flanked on either side by a base (N). The basesindicated by (N) may independently be a universal or degenerate base.Typically, the bases indicated by (N) are independently one of thecanonical bases. The bases indicated by (N) do not identify (or codefor) the complementary nucleotide it binds in the target sequence andare independent of the nucleic acid sequence of the (6-mer) sequence(b₁-b₂-b₃-b₆).

The sequencing probe depicted in FIG. 1 can be used in conjugation withthe sequencing methods of the present disclosure to sequence targetnucleic acids using only hybridization reactions, no covalent chemistry,enzymes or amplification is needed. To sequence all possible 6-mersequences in a target nucleic acid molecule, a total of 4096 sequencingprobes are needed (4{circumflex over ( )}6=4096).

FIG. 1 is exemplary for one configuration of a target binding domain ofthe sequence probe of the present disclosure. Table 1 provides severalother configurations of target binding domains of the presentdisclosure. One preferred target binding domain, called the “6 LNA”target binding domain, comprises 6 LNAs at positions b₁ to b₆ of thetarget binding domain. These 6 LNAs are flanked on either side by a base(N). As used herein, an (N) base can be a universal/degenerate base or acanonical base that is independent of the nucleic acid sequence of the(6-mer) sequence (b₁-b₂-b₃-b₄-b₅-b₆). In other words, while the basesb₁-b₂-b₃-b₄-b₅-b₆ may be specific to any given target sequence, the (N)bases can be a universal/degenerate base or composed of any of the fourcanonical bases that is not specific to the target dictated by basesb₁-b₂-b₃-b₄-b₅-b₆. For example, if the target sequence to beinterrogated is CAGGCATA bases b₁-b₂-b₃-b₄-b₅-b₆ of the target bindingdomain would be TCCGTA while each of the (N) bases of the target bindingdomain could independently be A, C, T or G such that a resulting targetbinding domain could have the sequence ATCCGTAG, TTCCGTAC, GTCCGTAG orany of the other 16 possible iterations. Alternatively, the two (N)bases could proceed the 6 LNAs. Alternatively still, the two (N) basescould follow the 6 LNAs.

TABLE 1 Target Binding Domain B1 B2 B3 B4 B5 B6 “6 LNA” N + + + + + + N“10mer” b b b b b b b b b b “Natural I” N N b b b b b b N N “Natural II”N b b b b b b N “2 LNA” N b b + + b b N N b + b b + b N N + b b b b + N“4 LNA” N + + b b + + N N + b + + b + N N b + + + + b N B = naturalbase; + = modified nucleotide or nucleotide analog (e.g. LNA); N =natural, universal or degenerate base

Table 1 also describes a “10 mer” target binding domain that comprises10 natural, target-specific bases.

Table 1 further describes the “Natural I” target binding domain thatcomprises 6 natural bases at positions b1 to b6. These 6 natural basesare flanked on either side by 2 (N) bases. Alternatively, all four (N)bases could proceed the 6 natural bases. Alternatively still, all four(N) bases could follow the 6 natural bases. Any number of the four (N)bases (i.e. 1, 2, 3 or 4) could proceed the 6 natural bases while theremaining (N) bases would follow the 6 natural bases.

Table 1 further describes the “Natural II” target binding domain thatcomprises 6 natural bases at positions b1 to b6. These 6 natural basesare flanked on either side by an (N) base. Alternatively, both (N) basescould proceed the 6 natural bases. Alternatively still, both (N) basescould follow the 6 natural bases.

Table 1 also describes a “2 LNA” target binding domain that comprises acombination of 2 LNAs and 4 natural bases at positions b1 to b6 of thetarget binding domain. The 2 LNAs and 4 natural bases can occur in anyorder. For example, the positions b3 and b4 can be LNAs while positionsb1, b2, b5 and b6 are natural bases. Bases b1 to b6 are flanked oneither side by a (N) base. Alternatively, bases b1 to b6 can beproceeded by two (N) bases. Alternatively still, bases b1 to b6 can befollowed by two (N) bases.

Table 1 further describes a “4 LNA” target binding domain that comprisesa combination of 4 LNAs and 2 natural bases at positions b1 to b6 of thetarget binding domain. The 4 LNAs and 2 natural bases can occur in anyorder. For example, the positions b2 to b5 can be LNAs while positionsb1 and b6 are natural bases. Bases b1 to b6 are flanked on either sideby a (N) base. Alternatively, bases b1 to b6 can be proceeded by two (N)bases. Alternatively still, bases b1 to b6 can be followed by two (N)bases.

The sequencing probe of the present disclosure comprises a syntheticbackbone. The target binding domain and the barcode domain are operablylinked. The target binding domain and barcode domain can be covalentlyattached, as part of one synthetic backbone. The target binding domainand barcode domain can be attached via a linker (e.g., nucleic acidlinker, chemical linker). The synthetic backbone can comprise anymaterial, e.g., polysaccharide, polynucleotide, polymer, plastic, fiber,peptide, peptide nucleic acid, or polypeptide. Preferably, the syntheticbackbone is rigid. The synthetic backbone can comprise a single-strandedDNA molecule. The backbone can comprise “DNA origami” of six DNA doublehelices (See, e.g., Lin et al, “Submicrometre geometrically encodedfluorescent barcodes self-assembled from DNA.” Nature Chemistry; 2012October; 4(10): 832-9). A barcode can be made of DNA origami tiles(Jungmann et al, “Multiplexed 3D cellular super-resolution imaging withDNA-PAINT and Exchange-PAINT”, Nature Methods, Vol. 11, No. 3, 2014).

The sequencing probe of the present disclosure can comprise a partiallydouble-stranded synthetic backbone. The sequencing probe can comprise asingle-stranded DNA synthetic backbone and a double-stranded DNA spacerbetween the target binding domain and the barcode domain. The sequencingprobe can comprise a single-stranded DNA synthetic backbone and apolymer-based spacer, with similar mechanical properties asdouble-stranded DNA, between the target binding domain and the barcodedomain. Typical polymer-based spacers include polyethylene glycol (PEG)type polymers.

The double-stranded DNA spacer can be from about 1 nucleotide to about100 nucleotides in length; from about 2 nucleotides to about 50nucleotides in length; from about 20 nucleotides to about 40 nucleotidesin length. Preferably, the double-stranded DNA spacer is about 36nucleotides in length.

One sequencing probe of the present disclosure, termed a “standardprobe” is illustrated in the left panel of FIG. 2. The standard probe ofFIG. 2 comprises a barcode domain covalently attached to the targetbinding domain, such that the target binding and barcode domains arepresent within the same single stranded oligonucleotide. In FIG. 2, leftpanel, a single stranded oligonucleotide binds to a stem oligonucleotideto create a 36 nucleotide long double-stranded spacer region called thestem. Using this architecture, each sequencing probe in a pool of probescan hybridize to the same stem sequence.

Another sequencing probe of the present disclosure, termed a “3 PartProbe” is illustrated in the right panel of FIG. 2. The 3 Part Probe ofFIG. 2 comprises a barcode domain that is attached to the target bindingdomain via a linker. In this example, the linker is a single strandedstem oligonucleotide that hybridizes to the single strandedoligonucleotide that contains the target binding domain and the singlestranded oligonucleotide that contains the barcode domain, creating a 36nucleotide long double stranded spacer region that bridges the barcodedomain (18 nucleotides) and target binding domain (18 nucleotides).Using this exemplary probe configuration, in order to prevent theexchange of barcode domains, each barcode can be designed such that ithybridizes to a unique stem sequence. Furthermore, each barcode domaincan also be hybridized to its corresponding stem oligonucleotide priorto pooling together different sequencing probes.

The barcode domain comprises a plurality of attachment positions, e.g.,one, two, three, four, five, six, seven, eight, nine, ten, or moreattachment positions. The number of attachment positions can be lessthan, equal to, or more than the number of nucleotides in the targetbinding domain. The target binding domain can comprise more nucleotidesthan number of attachment positions in the backbone domain, e.g., one,two, three, four, five, six, seven, eight, nine, ten, or morenucleotides. The target binding domain can comprise eight nucleotidesand the barcode domain comprises three attachment positions. The targetbinding domain can comprise ten nucleotides and the barcode domaincomprises three attachment positions

The length of the barcode domain is not limited as long as there issufficient space for at least three attachment positions, as describedbelow. The terms “attachment positions,” “positions” and “spots,” areused interchangeably herein. The terms “barcode domain” and “reportingdomain,” are used interchangeably herein.

Each attachment position in the barcode domain corresponds to twonucleotides (a dinucleotide) in the target binding domain and, thus, tothe complementary dinucleotide in the target nucleic acid that ishybridized to the dinucleotide in the target binding domain. As anon-limiting example, the first attachment position in the barcodedomain corresponds to the first and second nucleotides in the targetbinding domain (e.g., FIG. 1 where R1 is the first attachment positionin the barcode domain and R1 corresponds to dinucleotide b1 and b2 inthe target binding domain—which in turn identifies dinucleotides 1 and 2of the target nucleic acid); the second attachment position in thebarcode domain corresponds to the third and fourth nucleotides in thetarget binding domain (e.g., FIG. 1 where R2 is the second attachmentposition in the barcode domain and R2 corresponds to dinucleotide b3 andb4 in the target binding domain—which in turn identifies dinucleotides 3and 4 of the target nucleic acid); and the third attachment position inthe barcode domain corresponds to the fifth and sixth nucleotides in thetarget binding domain (e.g., FIG. 1 where R3 is the third attachmentposition in the barcode domain and R3 corresponds to dinucleotide b5 andb6 in the target binding domain—which in turn identifies dinucleotides 5and 6 of the target nucleic acid).

Each attachment position in the barcode domain comprises at least oneattachment region, e.g., one to 50, or more, attachment regions. Certainpositions in a barcode domain can have more attachment regions thanother positions (e.g., a first attachment position can have threeattachment regions whereas a second attachment position can have twoattachment positions); alternately, each position in a barcode domainhas the same number of attachment regions. Each attachment position inthe barcode domain can comprise one attachment region. Each attachmentposition in the barcode domain can comprise more than one attachmentregion. At least one of the at least three attachment positions in thebarcode domain can comprise a different number of attachment regionsthan the other two attachments positions in the barcode domain.

Each attachment region comprises at least one (i.e., one to fifty, e.g.,ten to thirty) copies of a nucleic acid sequence(s) capable of beingreversibly bound by a complementary nucleic acid molecule (e.g., DNA orRNA). The nucleic acid sequences of attachment regions at a singleattachment position can be identical; thus, the complementary nucleicacid molecules that bind those attachment regions are identical.Alternatively, the nucleic acid sequences of attachment regions at aposition are not identical; thus, the complementary nucleic acidmolecules that bind those attachment regions are not identical.

The nucleic acid sequence comprising each attachment region in a barcodedomain can be about 8 nucleotides to about 20 nucleotides in length. Thenucleic acid sequence comprising each attachment region in a barcodedomain can be about 12 or is about 14 nucleotides in length. Preferably,the nucleic acid sequence comprising each attachment region in a barcodedomain is about 14 nucleotides in length.

Each of the nucleic acids comprising each attachment region in a barcodedomain can independently be a canonical base or a modified nucleotide ornucleic acid analogue. At least one, at least two, at least three, atleast four, at least five, or at least six nucleotides in the attachmentregion in a barcode domain can be modified nucleotides or nucleotideanalogues. Typical ratios of modified nucleotides or nucleotideanalogues to canonical bases in a barcode domain are 1:2 to 1:8. Typicalmodified nucleotides or nucleic acid analogues useful in the attachmentregion in a barcode domain are isoguanine and isocytosine. The use ofmodified nucleotides or nucleotide analogues such as isoguanine andisocytosine, for example, can improve binding efficiency and accuracy ofthe reporter to the appropriate attachment region in a barcode domainwhile minimizing binding elsewhere, including to the target.

One or more attachment regions can be integral to a polynucleotidebackbone; that is, the backbone is a single polynucleotide and theattachment regions are parts of the single polynucleotide's sequence.One or more attachment regions can be linked to a modified monomer(e.g., modified nucleotide) in the synthetic backbone such that theattachment region branches from the synthetic backbone. An attachmentposition can comprise more than one attachment region, in which someattachment regions branch from the synthetic backbone and someattachment regions are integral to the synthetic backbone. At least oneattachment region in at least one attachment position can be integral tothe synthetic backbone. Each attachment region in each of the at leastthree attachment positions can be integral to the synthetic backbone. Atleast one attachment region in at least one attachment position canbranch from the synthetic backbone. Each attachment region in each ofthe at least three attachment positions can branch from the syntheticbackbone.

Each attachment position within a barcode domain corresponds to one ofsixteen dinucleotides i.e., either adenine-adenine,adenine-thymine/uracil, adenine-cytosine, adenine-guanine,thymine/uracil-adenine, thymine/uracil-thymine/uracil,thymine/uracil-cytosine, thymine/uracil-guanine, cytosine-adenine,cytosine-thymine/uracil, cytosine-cytosine, cytosine-guanine,guanine-adenine, guanine-thymine/uracil, guanine-cytosine orguanine-guanine. Thus, the one or more attachment regions located in asingle attachment position of a barcode domain correspond to one ofsixteen dinucleotides and comprise a nucleic acid sequence that isspecific to the dinucleotide to which the attachment region corresponds.Attachment regions located in different attachment positions of abarcode domain contain unique nucleic acid sequences even if thesepositions within the barcode domain correspond to the same dinucleotide.For example, given a sequencing probe of the present disclosure thatcontains a target binding domain with a hexamer that encodes thesequence A-G-A-G-A-C, the barcode domain of this sequencing probe wouldcontain three positions, with the first attachment positioncorresponding to an adenine-guanine dinucleotide, the second attachmentposition corresponding to an adenine-guanine dinucleotide and the thirdattachment position corresponding to an adenine-cytosine dinucleotide.The attachment regions located in position one of this example probewould comprise a nucleic acid sequence that is unique from the nucleicacid sequence of the attachment regions located in position two, eventhough both attachment position one and attachment position twocorrespond to the dinucleotide adenine-guanine. The sequences ofspecific attachment positions are designed and tested such that thecomplementary nucleic acid of a particular attachment position will notinteract with a different attachment position. Additionally, thenucleotide sequence of a complementary nucleic acid is not limited;preferably it lacks substantial homology (e.g., 50% to 99.9%) with aknown nucleotide sequence; this limits undesirable hybridization of acomplementary nucleic acid and a target nucleic acid.

FIG. 1 shows an illustration of one exemplary sequencing probe of thepresent disclosure comprising an exemplary barcode domain. The exemplarybarcode domain depicted in FIG. 1 comprises three attachment positions,R₁, R₂, and R₃. Each attachment position corresponds to a specificdinucleotide present within the 6-mer sequence (b₁ thru b₆) of thetarget binding domain. In this example, R₁ corresponds to positions b₁and b₂, R₂ corresponds to positions b₃ and b₄, and R₃ corresponds topositions b₅ and b₆. Thus, each position decodes a particulardinucleotide present in the 6-mer sequence of the target binding domain,allowing for the identification of the particular two bases (A, C, G orT) present in each particular dinucleotide.

In the exemplary barcode domain depicted in FIG. 1, each attachmentposition comprises a single attachment region that is integral to thesynthetic backbone. Each attachment region of the three attachmentpositions contains a specific nucleotide sequence that corresponds tothe particular dinucleotide that is encoded by each attachment position.For example, attachment position R₁ comprises an attachment region thathas a specific sequence that corresponds to the identity of thedinucleotide b₁-b₂.

The barcode domain can further comprise one or more binding regions. Thebarcode domain can comprise at least one single-stranded nucleic acidsequence adjacent or flanking at least one attachment position. Thebarcode domain can comprise at least two single-stranded nucleic acidsequences adjacent or flanking at least two attachment positions. Thebarcode domain can comprise at least three single-stranded nucleic acidsequences adjacent or flanking at least three attachment positions.These flanking portions are known as “Toe-Holds,” which can be used toaccelerate the rate of exchange of oligonucleotides hybridized adjacentto the Toe-Holds by providing additional binding sites forsingle-stranded oligonucleotides (e.g., “Toe-Hold” Probes; see, e.g.,Seeling et al., “Catalyzed Relaxation of a Metastable DNA Fuel”; J. Am.Chem. Soc. 2006, 128(37), pp 12211-12220).

Sequencing probes of the present disclosure can have overall lengths(including target binding domain, barcode domain, and any optionaldomains) of about 20 nanometers to about 50 nanometers. The sequencingprobe's backbone can be a polynucleotide molecule comprising about 120nucleotides.

A sequencing probe can comprise a cleavable linker modification. Anycleavable linker modification known to one of skill in the art can beutilized. Non-limiting examples of cleavable linker modificationsinclude, but are not limited to, UV-light cleavable linkers, reducingagent cleavable linkers and enzymatically cleavable linkers. An exampleof an enzymatically cleavable linker is the insertion of uracil forcleavage by the USER™ enzyme. The cleavable linker modification can belocated anywhere along the length of the sequencing probe, including,but not limited to, a region between the target binding domain and thebarcode domain. The right panel of FIG. 10 depicts exemplary cleavablelinker modifications that can be incorporated into the probes of thepresent disclosure.

Reporter Probes

A nucleic acid molecule that binds (e.g., hybridizes) to a complementarynucleic acid sequence within at least one attachment region within atleast one attachment position of a barcode domain of a sequencing probeof the present disclosure and comprises (directly or indirectly) adetectable label is referred to herein as a “reporter probe” or“reporter probe complex,” these terms are used interchangeably herein.The reporter probe can be DNA, RNA or PNA. Preferably, the reporterprobe is DNA.

A reporter probe can comprise at least two domains, a first domaincapable of binding at least one first complementary nucleic acidmolecule and a second domain capable of binding a first detectable labeland at least a second detectable label. FIG. 3 shows a schematic of anexemplary reporter probe of the present disclosure bound to the firstattachment position of a barcode domain of an exemplary sequencingprobe. In FIG. 3, the first domain of the reporter probe (shown inhatched maroon) binds a complementary nucleic acid sequence withinattachment position R₁ of the barcode domain and the second domain ofthe reporter probe (shown in gray) is bound to two detectable labels(one green label, one red label).

Alternatively, the reporter probe can comprise at least two domains, afirst domain capable of binding at least one first complementary nucleicacid molecule and a second domain capable of binding at least one secondcomplementary nucleic acid molecule. The at least one first and at leastone second complementary nucleic acid molecules can be different (havedifferent nucleic acid sequences).

A “primary nucleic acid molecule” is a reporter probe comprising atleast two domains, a first domain capable of binding (e.g. hybridizing)to a complementary nucleic acid sequence within at least one attachmentregion within at least one attachment position of a barcode domain of asequencing probe and a second domain capable of binding (e.g.hybridizing) to at least one additional complementary nucleic acid. Aprimary nucleic acid molecule can directly bind the complementarynucleic acid sequence within the at least one attachment region withinthe at least one attachment position of a barcode domain of a sequencingprobe. A primary nucleic acid molecule can indirectly bind thecomplementary nucleic acid sequence within the at least one attachmentregion within the at least one attachment position of a barcode domainof a sequencing probe via a nucleic acid linker. The primary nucleicacid molecule can comprise a cleavable linker. The cleavable linker canbe located between the first domain and the second domain. Preferably,the cleavable linker is photo-cleavable.

Each of the nucleic acids comprising the first domain of a primarynucleic acid molecule can independently be a canonical base or amodified nucleotide or nucleic acid analogue. At least one, two, atleast three, at least four, at least five, or at least six nucleotidesin the first domain of a primary nucleic acid molecule can be modifiednucleotides or nucleotide analogues. Typical ratios of modifiednucleotides or nucleotide analogues to canonical bases in a barcodedomain are 1:2 to 1:8. Typical modified nucleotides or nucleic acidanalogues useful in the first domain of a primary nucleic acid moleculeare isoguanine and isocytosine. The use of modified nucleotides ornucleotide analogues such as isoguanine and isocytosine, for example,can improve binding efficiency and accuracy of the first domain of aprimary nucleic acid molecule to the appropriate complementary nucleicacid sequence within at least one attachment region within at least oneattachment position of a barcode domain of a sequencing probe whileminimizing binding elsewhere, including to the target.

The at least one additional complementary nucleic acid that binds theprimary nucleic acid molecule is referred to herein as a “secondarynucleic molecule.” The primary nucleic acid molecule can bind (e.g.,hybridize) to at least one, at least two, at least three, at least four,at least five, or more secondary nucleic acid molecules. Preferably, theprimary nucleic acid molecule binds (e.g., hybridizes) to four secondarynucleic acid molecules.

A secondary nucleic acid molecule comprises at least two domains, afirst domain capable of binding (e.g. hybridizing) to at least onecomplementary sequence in at least one primary nucleic acid molecule anda second domain capable of binding (e.g. hybridizing) to (a) a firstdetectable label and an at least second detectable label; (b) to atleast one additional complementary nucleic acid; or (c) a combinationthereof. The secondary nucleic acid molecule can comprise a cleavablelinker. The cleavable linker can be located between the first domain andthe second domain. Preferably, the cleavable linker is photo-cleavable.

Each of the nucleic acids comprising the first domain of a secondarynucleic acid molecule can independently be a canonical base or amodified nucleotide or nucleic acid analogue. At least one, two, atleast three, at least four, at least five, or at least six nucleotidesin the first domain of a secondary nucleic acid molecule can be modifiednucleotides or nucleotide analogues. Typical ratios of modifiednucleotides or nucleotide analogues to canonical bases in a barcodedomain are 1:2 to 1:8. Typical modified nucleotides or nucleic acidanalogues useful in the first domain of a secondary nucleic acidmolecule are isoguanine and isocytosine. The use of modified nucleotidesor nucleotide analogues such as isoguanine and isocytosine, for example,can improve binding efficiency and accuracy of the first domain of asecondary nucleic acid molecule to the appropriate complementary nucleicacid sequence within the second domain of a primary nucleic acidmolecule while minimizing binding elsewhere.

The at least one additional complementary nucleic acid that binds thesecondary nucleic acid molecule is referred to herein as a “tertiarynucleic molecule.” The secondary nucleic acid molecule can bind (e.g.,hybridize) to at least one, at least two, at least three, at least four,at least five, at least six, at least seven, or more tertiary nucleicacid molecules. Preferably, the at least one secondary nucleic acidmolecule binds (e.g., hybridizes) to one tertiary nucleic acid molecule.

A tertiary nucleic acid molecule comprises at least two domains, a firstdomain capable of binding (e.g. hybridizing) to at least onecomplementary sequence in at least one secondary nucleic acid moleculeand a second domain capable of binding (e.g. hybridizing) to a firstdetectable label and an at least second detectable label. Alternatively,the second domain can include the first detectable label and an at leastsecond detectable label via direct or indirect attachment of the labelsduring oligonucleotide synthesis using, for example, phosphoroamidite orNHS chemistry. The tertiary nucleic acid molecule can comprise acleavable linker. The cleavable linker can be located between the firstdomain and the second domain. Preferably, the cleavable linker isphoto-cleavable.

Each of the nucleic acids comprising the first domain of a tertiarynucleic acid molecule can independently be a canonical base or amodified nucleotide or nucleic acid analogue. At least one, two, atleast three, at least four, at least five, or at least six nucleotidesin the first domain of a tertiary nucleic acid can be modifiednucleotides or nucleotide analogues. Typical ratios of modifiednucleotides or nucleotide analogues to canonical bases in a first domainof a tertiary nucleic acid molecule are 1:2 to 1:8. Typical modifiednucleotides or nucleic acid analogues useful in the first domain of atertiary nucleic acid molecule are isoguanine and isocytosine. The useof modified nucleotides or nucleotide analogues such as isoguanine andisocytosine, for example, can improve binding efficiency and accuracy ofthe first domain of a tertiary nucleic acid molecule to the appropriatecomplementary nucleic acid sequence within the second domain of a secondnucleic acid molecule while minimizing binding elsewhere.

Reporter probes are bound to a first detectable label and an at leastsecond detectable label to create a dual color combination. This dualcombination of fluorescent dyes can include a duplicity of a singlecolor, e.g. blue-blue. As used herein, the term “label” includes asingle moiety capable to producing a detectable signal or multiplemoieties capable of producing the same or substantially the samedetectable signal. For example, a label includes a single yellowfluorescent dye such as ALEXA FLUOR™ 532 or multiple yellow fluorescentdyes such as ALEXA FLUOR™ 532.

The reporter probes can bind to a first detectable label and an at leastsecond detectable label, in which each detectable label is one of fourfluorescent dyes: blue (B); green (G); yellow (Y); and red (R). The useof these four dyes creates 10 possible dual color combinations BB; BG;BR; BY; GG; GR; GY; RR; RY; or YY. In some aspects, reporter probes ofthe present disclosure are labeled with one of 8 possible colorcombinations: BB; BG; BR; BY; GG; GR; GY; or YY as depicted in FIG. 3.The detectable label and an at least second detectable label can havethe same emission spectrum or can have a different emission spectra.

In aspects comprising a sequencing probe and a primary nucleic acidmolecule, the present disclosure provides a sequencing probe comprisinga target binding domain and a barcode domain; wherein the target bindingdomain comprises at least eight nucleotides and is capable of binding atarget nucleic acid, wherein at least six nucleotides in the targetbinding domain are capable of identifying a corresponding(complementary) nucleotide in the target nucleic acid molecule andwherein at least two nucleotides in the target binding domain do notidentify a corresponding nucleotide in the target nucleic acid molecule;wherein at least one, or at least two nucleotides, of the at least sixnucleotides in the target binding domain are modified nucleotides ornucleotide analogues; wherein the barcode domain comprises a syntheticbackbone, the barcode domain comprising at least three attachmentpositions, each attachment position comprising at least one attachmentregion comprising at least one nucleic acid sequence bound by at leastone complementary primary nucleic acid molecule, wherein thecomplementary primary nucleic acid molecule comprises a first detectablelabel and at least a second detectable label, wherein each attachmentposition of the at least three attachment positions corresponds to twonucleotides of the at least six nucleotides in the target binding domainand each of the at least three attachment positions have a differentnucleic acid sequence, and wherein the at least first detectable labeland at least second detectable label of each complementary primarynucleic acid molecule bound to each position of the at least threeattachment positions determines the position and identity of thecorresponding two nucleotides of the at least six nucleotides in thetarget nucleic acid that is bound by the target binding domain. The atleast two nucleotides in the target binding domain that do not identifya corresponding nucleotide in the target nucleic acid molecule can beany of the four canonical bases that is not specific to the targetdictated by the at least six nucleotides in the target binding domain oruniversal or degenerate bases.

In aspects comprising a sequencing probe and a primary nucleic acidmolecule, the present disclosure also provides a sequencing probecomprising a target binding domain and a barcode domain; wherein thetarget binding domain comprises at least ten nucleotides and is capableof binding a target nucleic acid, wherein at least six nucleotides inthe target binding domain are capable of identifying a corresponding(complementary) nucleotide in the target nucleic acid molecule andwherein at least four nucleotides in the target binding domain do notidentify a corresponding nucleotide in the target nucleic acid molecule;wherein the barcode domain comprises a synthetic backbone, the barcodedomain comprising at least three attachment positions, each attachmentposition comprising at least one attachment region comprising at leastone nucleic acid sequence bound by at least one complementary primarynucleic acid molecule, wherein the complementary primary nucleic acidmolecule comprises at first detectable label and at least a seconddetectable label, wherein each attachment position of the at least threeattachment positions corresponds to two nucleotides of the at least sixnucleotides in the target binding domain and each of the at least threeattachment positions have a different nucleic acid sequence, wherein theat least first detectable label and at least second detectable label ofeach complementary primary nucleic acid molecule bound to each positionof the at least three attachment positions determines the position andidentity of the corresponding two nucleotides of the at least sixnucleotides in the target nucleic acid that is bound by the targetbinding domain.

In aspects comprising a sequencing probe, a primary nucleic acidmolecule and a secondary nucleic acid molecule, the present disclosureprovides a sequencing probe comprising a target binding domain and abarcode domain; wherein the target binding domain comprises at leasteight nucleotides and is capable of binding a target nucleic acid,wherein at least six nucleotides in the target binding domain arecapable of identifying a corresponding (complementary) nucleotide in thetarget nucleic acid molecule and wherein at least two nucleotides in thetarget binding domain do not identify a corresponding nucleotide in thetarget nucleic acid molecule; wherein at least one, or at least twonucleotides, of the at least six nucleotides in the target bindingdomain are modified nucleotides or nucleotide analogues and wherein theat least one, or at least two nucleotides in the target binding domainare any of the four canonical bases that is not specific to the targetdictated by the other bases in the target binding domain or universal ordegenerate bases; wherein the barcode domain comprises a syntheticbackbone, the barcode domain comprising at least three attachmentpositions, each attachment position comprising at least one attachmentregion comprising at least one nucleic acid sequence bound by at leastone complementary primary nucleic acid molecule, wherein thecomplementary primary nucleic acid molecule is further bound by at leastone complementary secondary nucleic acid molecule comprising at firstdetectable label and at least a second detectable label, wherein eachattachment position of the at least three attachment positionscorresponds to two nucleotides of the at least six nucleotides in thetarget binding domain and each of the at least three attachmentpositions have a different nucleic acid sequence, and wherein the atleast first detectable label and at least second detectable label ofeach complementary secondary nucleic acid molecule bound to eachposition of the at least three attachment positions determines theposition and identity of the corresponding two nucleotides of the atleast six nucleotides in the target nucleic acid that is bound by thetarget binding domain.

In aspects comprising a sequencing probe, a primary nucleic acidmolecule and a secondary nucleic acid molecule, the present disclosurealso provides a sequencing probe comprising a target binding domain anda barcode domain; wherein the target binding domain comprises at leastten nucleotides and is capable of binding a target nucleic acid, whereinat least six nucleotides in the target binding domain are capable ofidentifying a corresponding (complementary) nucleotide in the targetnucleic acid molecule and wherein at least four nucleotides in thetarget binding domain do not identify a corresponding nucleotide in thetarget nucleic acid molecule; wherein the barcode domain comprises asynthetic backbone, the barcode domain comprising at least threeattachment positions, each attachment position comprising at least oneattachment region comprising at least one nucleic acid sequence bound byat least one complementary primary nucleic acid molecule, wherein thecomplementary primary nucleic acid molecule is further bound by at leastone complementary secondary nucleic acid molecule comprising at firstdetectable label and at least a second detectable label, wherein eachattachment position of the at least three attachment positionscorresponds to two nucleotides of the at least six nucleotides in thetarget binding domain and each of the at least three attachmentpositions have a different nucleic acid sequence, wherein the at leastfirst detectable label and at least second detectable label of eachcomplementary secondary nucleic acid molecule bound to each position ofthe at least three attachment positions determines the position andidentity of the corresponding two nucleotides of the at least sixnucleotides in the target nucleic acid that is bound by the targetbinding domain.

In aspects comprising a sequencing probe, a primary nucleic acidmolecule, a secondary nucleic acid molecule and a tertiary nucleic acidmolecule, the present disclosure provides a sequencing probe comprisinga target binding domain and a barcode domain; wherein the target bindingdomain comprises at least eight nucleotides and is capable of binding atarget nucleic acid, wherein at least six nucleotides in the targetbinding domain are capable of identifying a corresponding(complementary) nucleotide in the target nucleic acid molecule andwherein at least two nucleotides in the target binding domain do notidentify a corresponding nucleotide in the target nucleic acid molecule;wherein at least one, or at least two nucleotides, of the at least sixnucleotides in the target binding domain are modified nucleotides ornucleotide analogues and wherein the least two nucleotides in the targetbinding domain that do not identify a corresponding nucleotide in thetarget nucleic acid molecule can be any of the four canonical bases thatis not specific to the target dictated by the at least six nucleotidesin the target binding domain or universal or universal or degeneratebases; wherein the barcode domain comprises a synthetic backbone, thebarcode domain comprising at least three attachment positions, eachattachment position comprising at least one attachment region comprisingat least one nucleic acid sequence bound by at least one complementaryprimary nucleic acid molecule, wherein the complementary primary nucleicacid molecule is further bound by at least one complementary secondarynucleic acid molecule, and wherein the at least one complementarysecondary nucleic acid molecule is further bound by at least onecomplementary tertiary nucleic acid molecule comprising at firstdetectable label and at least a second detectable label, wherein eachattachment position of the at least three attachment positionscorresponds to two nucleotides of the at least six nucleotides in thetarget binding domain and each of the at least three attachmentpositions have a different nucleic acid sequence, and wherein the atleast first detectable label and at least second detectable label ofeach complementary tertiary nucleic acid molecule bound to each positionof the at least three attachment positions determines the position andidentity of the corresponding two nucleotides of the at least sixnucleotides in the target nucleic acid that is bound by the targetbinding domain.

In aspects comprising a sequencing probe, a primary nucleic acidmolecule, a secondary nucleic acid molecule and a tertiary nucleic acidmolecule, the present disclosure also provides a sequencing probecomprising a target binding domain and a barcode domain; wherein thetarget binding domain comprises at least ten nucleotides and is capableof binding a target nucleic acid, wherein at least six nucleotides inthe target binding domain are capable of identifying a corresponding(complementary) nucleotide in the target nucleic acid molecule andwherein at least four nucleotides in the target binding domain do notidentify a corresponding nucleotide in the target nucleic acid molecule;wherein the barcode domain comprises a synthetic backbone, the barcodedomain comprising at least three attachment positions, each attachmentposition comprising at least one attachment region comprising at leastone nucleic acid sequence bound by at least one complementary primarynucleic acid molecule, wherein the complementary primary nucleic acidmolecule is further bound by at least one complementary secondarynucleic acid molecule, and wherein the at least one complementarysecondary nucleic acid molecule is further bound by at least onecomplementary tertiary nucleic acid molecule comprising at firstdetectable label and at least a second detectable label, wherein eachattachment position of the at least three attachment positionscorresponds to two nucleotides of the at least six nucleotides in thetarget binding domain and each of the at least three attachmentpositions have a different nucleic acid sequence, wherein the at leastfirst detectable label and at least second detectable label of eachcomplementary tertiary nucleic acid molecule bound to each position ofthe at least three attachment positions determines the position andidentity of the corresponding two nucleotides of the at least sixnucleotides in the target nucleic acid that is bound by the targetbinding domain.

The present disclosure also provides sequencing probes and reporterprobes having detectable labels on both a secondary nucleic acidmolecule and a tertiary nucleic acid molecule. For example, a secondarynucleic acid molecule can bind a primary nucleic acid molecule and thesecondary nucleic acid molecule can comprise both a first detectablelabel and an at least second detectable label and also be bound to atleast one tertiary molecule comprising a first detectable label and anat least second detectable label. The first and at least seconddetectable labels located on the secondary nucleic acid molecule canhave the same emission spectra or can have different emission spectra.The first and at least second detectable labels located on the tertiarynucleic acid molecule can have the same emission spectra or can havedifferent emission spectra. The emission spectra of the detectablelabels on the secondary nucleic acid molecule can be the same or can bedifferent than the emission spectra of the detectable labels on thetertiary nucleic acid molecule.

FIG. 4 is an illustrative schematic of an exemplary reporter probe ofthe present disclosure that comprises an exemplary primary nucleic acidmolecule, secondary nucleic acid molecule and tertiary nucleic acidmolecule. At the 3′ end, the primary nucleic acid comprises a firstdomain, wherein the first domain comprises a twelve nucleotide sequencethat hybridizes to a complementary attachment region within anattachment position of a sequencing probe barcode domain. At the 5′ endis a second domain that is hybridized to six secondary nucleic acidmolecules. The exemplary secondary nucleic acid molecules depicted inturn comprise a first domain in the 5′ end that hybridizes to theprimary nucleic acid molecule and a domain that in the 3′ portion thathybridizes to five tertiary nucleic acid molecules.

A tertiary nucleic acid molecule comprises at least two domains. Thefirst domain is capable of binding to a secondary nucleic acid molecule.The second domain of a tertiary nucleic acid is capable of binding to afirst detectable label and at least second detectable label. The seconddomain of a tertiary nucleic acid can be bound to the first detectablelabel and at least second detectable label by the direct incorporationof one or more fluorescently-labeled nucleotide monomers into thesequence of the second domain of the tertiary nucleic acid. The seconddomain of the secondary nucleic acid molecule can be bound by the firstdetectable label and at least second detectable label by hybridizingshort polynucleotides that are labeled to the second domain of thesecondary nucleic acid. These short polynucleotides, called“labeled-oligos,” can be labeled by direct incorporation offluorescently-labeled nucleotide monomers or by other methods oflabeling nucleic acids that are known to one of skill in that art. Theexemplary tertiary nucleic acid molecules depicted in FIG. 4, which maybe considered “labeled oligos” comprise a first domain that hybridizesto a secondary nucleic acid molecule and a second domain that isfluorescently labeled by indirect attachment of the labels duringoligonucleotide synthesis using, for example, NHS chemistry orincorporation of one or more fluorescently-labeled nucleotide monomersduring the synthesis of the tertiary nucleic acid molecule. Thelabeled-oligos can be DNA, RNA or PNA.

In alternative aspects, the second domain of a secondary nucleic acid iscapable of binding to a first detectable label and at least seconddetectable label. The second domain of the secondary nucleic acid can bebound to the first detectable label and at least second detectable labelby the direct incorporation of one or more fluorescently-labelednucleotide monomers into the sequence of the second domain of thesecondary nucleic acid. The second domain of the secondary nucleic acidmolecule can be bound by the first detectable label and at least seconddetectable label by hybridizing short polynucleotides that are labeledto the second domain of the secondary nucleic acid. These shortpolynucleotides, called labeled-oligos, can be labeled by directincorporation of fluorescently-labeled nucleotide monomers or by othermethods of labeling nucleic acids that are known to one of skill in thatart.

A primary nucleic acid molecule can comprise about 100, about 95, about90, about 85, about 80 or about 75 nucleotides. A primary nucleic acidmolecule can comprise about 100 to about 80 nucleotides. A primarynucleic acid molecule can comprise about 90 nucleotides. A secondarynucleic acid molecule can comprise about 90, about 85, about 80, about75 or about 70 nucleotides. A secondary nucleic acid molecule cancomprise about 90 to about 80 nucleotides. A secondary nucleic acidmolecule can comprise about 87 nucleotides. A secondary nucleic acidmolecule can comprise about 25, about 20, about 15, or about 10nucleotides. A tertiary nucleic acid molecule can comprise about 20 toabout 10 nucleotides. A tertiary nucleic acid molecule can compriseabout 15 nucleotides.

Reporter probes of the present disclosure can be of various designs. Forexample, a primary nucleic acid molecule can be hybridized to at leastone (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) secondary nucleicacid molecules. Each secondary nucleic acid molecule can be hybridizedto at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) tertiarynucleic acid molecules. To create a reporter probe that is labeled witha particular dual color combination, the reporter probe is designed suchthat the probe comprises secondary nucleic acid molecules, tertiarynucleic acid molecules, labeled-oligos or any combination of secondarynucleic acid molecules, tertiary nucleic acid molecules andlabeled-oligos that are labeled with each color of the particular dualcolor combination. For example, FIG. 4 depicts a reporter probe of thepresent disclosure that comprises 30 total dyes, with 15 dyes for color1 and 15 dyes for color 2. To prevent color-swapping or crosshybridization between different fluorescent dyes, each tertiary nucleicacid or labeled-oligo that is bound to a specific label or fluorescentdye comprises a unique nucleotide sequence.

FIG. 5 depicts four exemplary reporter probe designs of the presentdisclosure. The top left panel of FIG. 5 depicts a 5×5 reporter probe. A5×5 reporter probe comprises a primary nucleic acid, wherein the primarynucleic acid comprises a first domain of 12 nucleotides. The primarynucleic acid also comprises a second domain, wherein the second domaincomprises a nucleotide sequence that can be hybridized to 5 secondarynucleic acid molecules. Each secondary nucleic acid comprises anucleotide sequence such that 5 tertiary nucleic acids that are bound bydetectable labels can hybridize to each secondary nucleic acid.

The top right panel of FIG. 5 depicts a 4×3 reporter probe. A 4×3reporter probe comprises a primary nucleic acid, wherein the primarynucleic acid comprises a first domain of 12 nucleotides. The primarynucleic acid also comprises a second domain, wherein the second domaincomprises a nucleotide sequence that can be hybridized to 4 secondarynucleic acid molecules. Each secondary nucleic acid comprises anucleotide sequence such that 3 tertiary nucleic acids that are bound todetectable labels can hybridize to each secondary nucleic acid.

The bottom left panel of FIG. 5 depicts a 3×4 reporter probe. A 3×4reporter probe comprises a primary nucleic acid, wherein the primarynucleic acid comprises a first domain of 12 nucleotides. The primarynucleic acid also comprises a second domain, wherein the second domaincomprises a nucleotide sequence that can be hybridized to 3 secondarynucleic acid molecules. Each secondary nucleic acid comprises anucleotide sequence such that 4 tertiary nucleic acids that are bound todetectable labels can hybridize to each secondary nucleic acid.

The bottom right panel of FIG. 5 depicts a Spacer 3×4 reporter probe. ASpacer 3×4 reporter probe comprises a primary nucleic acid, wherein theprimary nucleic acid comprises a first domain of 12 nucleotides. Locatedbetween the first domain and second domain of the primary nucleic acidis a spacer region consisting of 20 to 40 nucleotides. The spacer isidentified as 20 to 40 nucleotides long; however, the length of a spaceris non-limiting and it can be shorter than 20 nucleotides or longer than40 nucleotides. The second domain of the primary nucleic acid comprisesa nucleotide sequence that can hybridize to 3 secondary nucleic acidmolecules. Each secondary nucleic acid comprises a nucleotide sequencesuch that 4 tertiary nucleic acids that are bound to detectable labelscan hybridize to each secondary nucleic acid.

In FIG. 5, each primary nucleic acid comprises a first domain that is 12nucleotides long. However, the length of the first domain of a primarynucleic acid is non-limited and can be less than 12 or more than 12nucleotides. Preferably, the first domain of a primary nucleic acid is14 nucleotides.

Any of the features of a specific reporter probe design depicted in aparticular panel of FIG. 5 can be combined with any of the features of areporter probe design depicted in a different panel of FIG. 5 ordescribed elsewhere herein. For example, a 5×5 reporter probe can bemodified to contain a spacer region of approximately 20 to 40nucleotides between the complementary nucleic and the primary nucleicacid. In another example, a 4×3 reporter probe can be modified such thatthe 4 secondary nucleic acids comprise a nucleotide sequence that allows5 tertiary nucleic acids that are bound to detectable labels tohybridize to each secondary nucleic acid, thereby creating a 4×5reporter probe.

Referring to FIG. 5, a 5×5 reporter contains more fluorescent labels(25) than a 4×3 reporter (12) and therefore the fluorescent intensity ofthe 5×5 reporter will be greater. The fluorescence detected in any givenfield of view FOV is a function a variety of variable including thefluorescent intensity of the given reporter probes and the number ofoptionally bound target molecules within that FOV. The number ofoptionally bound target molecules per field of view (FOV) can be from 1to 2.5 million targets per FOV. Typical numbers of bound targetmolecules per FOV are 20,000 to 40,000, 220,000 to 440,000 or 1 millionto 2 million target molecules. Typical FOVs are 0.05 mm² to 1 mm².Further examples of typical FOVs are 0.05 mm² to 0.65 mm².

FIG. 6 shows reporter probe designs in which the secondary nucleic acidmolecules comprise “extra-handles” that are not hybridized to a tertiarynucleic acid molecule and are distal to the primary nucleic acidmolecule. In FIG. 6, each “extra-handle” is 12 nucleotides long (“12mer”); however, their lengths are non-limited and can be less than 12 ormore than 12 nucleotides. The “extra-handles” can each comprise thenucleotide sequence of the first domain of the primary nucleic acidmolecule to which the secondary nucleic acid molecule is hybridized.Thus, when a reporter probe comprises “extra-handles”, the reporterprobe can hybridize to a sequencing probe either via the first domain ofthe primary nucleic acid molecule or via an “extra-handle.” Accordingly,the likelihood that a reporter probe binds to a sequencing probe isincreased. The “extra-handle” design can also improve hybridizationkinetics. Without being bound by any theory, the “extra-handles” canincrease the effective concentration of the reporter probe'scomplementary nucleic acid. A 5×4 “extra-handles” reporter probe isexpected to yield approximately 4750 fluorescent counts per standardFOV. A 5×3 “extra-handles” reporter probe, a 4×4 “extra-handles”reporter probe, a 4×3 “extra-handles” reporter probe and a 3×4“extra-handles” reporter probe are all expected to yield approximately6000 fluorescent counts per standard FOV. The exemplary reporter probedesigns depicted in FIG. 5 can also be modified to include“extra-handles”.

Individual secondary nucleic acid molecules of a reporter probe canhybridize to tertiary nucleic acid molecules that are all labeled withthe same detectable label. For example, the left panel of FIG. 7 depictsa “5×6” reporter probe. A 5×6 reporter probe comprises one primarynucleic acid that comprises a second domain, wherein the second domaincomprises a nucleotide sequence hybridized to 6 secondary nucleic acidmolecules. Each secondary nucleic acid comprises a nucleotide sequencesuch that 5 tertiary nucleic acid molecules that are bound to detectablelabels hybridized to each secondary nucleic acid. Each of the 5 tertiarynucleic acid molecules that bind to a particular secondary nucleic acidmolecule are labeled with the same detectable label. Three of thesecondary nucleic acid molecules bind to tertiary nucleic acid moleculeslabeled with a yellow fluorescent dye and the other three secondarynucleic acid bind to tertiary nucleic acid molecules labeled with a redfluorescent dye, for example.

Individual secondary nucleic acid molecules of a reporter probe canhybridize to tertiary nucleic acid molecules that are labeled withdifferent detectable labels. For example, the middle panel of FIG. 7depicts a “3×2×6” reporter probe design. A “3×2×6” reporter probecomprises one primary nucleic acid that comprises a second domain,wherein the second domain comprises a nucleotide sequence hybridized to6 secondary nucleic acid molecules. Each secondary nucleic acidcomprises a nucleotide sequence such that 5 tertiary nucleic acids thatare bound to detectable labels hybridized to each secondary nucleicacid. Each secondary nucleic acid binds to both tertiary nucleic acidmolecules labeled with a yellow fluorescent dye and to tertiary nucleicacid molecules labeled with a red fluorescent dye. In this specificexample, three secondary nucleic acid molecules bind two red and threeyellow tertiary nucleic acid molecules, while the other three secondarynucleic acid molecules bind two red and three yellow tertiary nucleicacid molecules. Each secondary nucleic acid molecule can bind to anynumber of tertiary nucleic acid molecules bound by different detectablelabels. In the middle panel of FIG. 7, the tertiary nucleic acidmolecules bound to an individual secondary nucleic acid molecule arearranged such that the colors of the label alternate (i.e.red-yellow-red-yellow-red or yellow-red-yellow-red-yellow).

In any of the described reporter probe designs, tertiary nucleic acidslabeled with different detectable labels can be arranged in any orderalong the secondary nucleic acid. For example, the right panel of FIG. 7depicts a “Fret resistant 3×2×6” reporter probe that is similar to the3×2×6 reporter probe design except in the arrangement (e.g., linearorder or grouping) of red and yellow tertiary nucleic acid moleculesalong each secondary nucleic acid molecule.

FIG. 8 depicts more exemplary reporter probe designs of the presentdisclosure that include individual secondary nucleic acid molecules thatbind to varying tertiary nucleic acid molecules. The left panel depictsa “6×1×4.5” reporter probe that comprises one primary nucleic acidmolecule, wherein the primary nucleic acid molecule comprises a seconddomain, wherein the second domain comprises a nucleotide sequencehybridized to six secondary nucleic acid molecules. Each secondarynucleic acid molecule is hybridized to five tertiary nucleic acidmolecules. Four of the five tertiary nucleic acid molecules thathybridize to each secondary nucleic acid molecule are directly labeledwith the same color detectable label. The fifth tertiary nucleic acid,denoted as the branching tertiary nucleic acid, is bound to 5labeled-oligos of the other color of the dual color combination. Of thesix secondary nucleic acids, three of them bind to a branching tertiarynucleic acid labeled with one color of the dual color combination (inthis example red), while the other three secondary nucleic acids bind toa branching tertiary nucleic acid labeled with the other color of thedual color combination (in this example yellow). In total, the 6×1×4.5reporter probe is labeled with 54 total dyes, 27 dyes for each color.The middle panel of FIG. 8 depicts a “4×1×4.5” reporter probe thatshares the same overall architecture as the 6×1×4.5 reporter probe,except that the primary nucleic acid of the 4×1×4.5 reporter probe bindsonly 4 secondary nucleic acids, such that there are a total of 36 dyes,18 for each color.

A reporter probe can comprise the same number of dyes for each color ofthe dual color combination. A reporter probe can comprise a differentnumber of dyes for each color of the dual color combination. Theselection as to which color has more dyes within a reporter probe can bemade on the basis of the energy level of light that the two dyes absorb.For example, the right panel of FIG. 8 depicts a “5×5 energy optimized”reporter probe design. This reporter probe design comprises 15 yellowdyes (which are higher energy) and 10 red dyes (which are lower energy).In this example, the 15 yellow dyes can constitute a first label and the10 red dyes can constitute a second label.

A detectable moiety, label or reporter can be bound to a secondarynucleic acid molecule, a tertiary nucleic acid molecule or to alabeled-oligo in a variety of ways, including the direct or indirectattachment of a detectable moiety such as a fluorescent moiety,colorimetric moiety and the like. One of skill in the art can consultreferences directed to labeling nucleic acids. Examples of fluorescentmoieties include, but are not limited to, yellow fluorescent protein(YFP), green fluorescent protein (GFP), cyan fluorescent protein (CFP),red fluorescent protein (RFP), umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, cyanines,dansyl chloride, phycocyanin, phycoerythrin and the like.

Fluorescent labels and their attachment to nucleotides and/oroligonucleotides are described in many reviews, including Haugland,Handbook of Fluorescent Probes and Research Chemicals, Ninth Edition(Molecular Probes, Inc., Eugene, 2002); Keller and Manak, DNA Probes,2nd Edition (Stockton Press, New York, 1993); Eckstein, editor,Oligonucleotides and Analogues: A Practical Approach (IRL Press, Oxford,1991); and Wetmur, Critical Reviews in Biochemistry and MolecularBiology, 26:227-259 (1991). Particular methodologies applicable to thedisclosure are disclosed in the following sample of references: U.S.Pat. Nos. 4,757,141; 5,151,507; and 5,091,519. One or more fluorescentdyes can be used as labels for labeled target sequences, e.g., asdisclosed by U.S. Pat. No. 5,188,934 (4,7-dichlorofluorescein dyes);U.S. Pat. No. 5,366,860 (spectrally resolvable rhodamine dyes); U.S.Pat. No. 5,847,162 (4,7-dichlororhodamine dyes); U.S. Pat. No. 4,318,846(ether-substituted fluorescein dyes); U.S. Pat. No. 5,800,996 (energytransfer dyes); Lee et al. U.S. Pat. No. 5,066,580 (xanthine dyes); U.S.Pat. No. 5,688,648 (energy transfer dyes); and the like. Labelling canalso be carried out with quantum dots, as disclosed in the followingpatents and patent publications: U.S. Pat. Nos. 6,322,901; 6,576,291;6,423,551; 6,251,303; 6,319,426; 6,426,513; 6,444,143; 5,990,479;6,207,392; 2002/0045045; and 2003/0017264. As used herein, the term“fluorescent label” comprises a signaling moiety that conveysinformation through the fluorescent absorption and/or emissionproperties of one or more molecules. Such fluorescent properties includefluorescence intensity, fluorescence lifetime, emission spectrumcharacteristics, energy transfer, and the like.

Commercially available fluorescent nucleotide analogues readilyincorporated into nucleotide and/or oligonucleotide sequences include,but are not limited to, Cy3-dCTP, Cy3-dUTP, Cy5-dCTP, Cy5-dUTP (AmershamBiosciences, Piscataway, N.J.), fluorescein-12-dUTP,tetramethylrhodamine-6-dUTP, TEXAS RED™-5-dUTP, CASCADE BLUE™-7-dUTP,BODIPY TMFL-14-dUTP, BODIPY TMR-14-dUTP, BODIPY TMTR-14-dUTP, RHODAMINEGREEN™-5-dUTP, OREGON GREENR™ 488-5-dUTP, TEXAS RED™-12-dUTP, BODIPY™630/650-14-dUTP, BODIPY™ 650/665-14-dUTP, ALEXA FLUOR™ 488-5-dUTP, ALEXAFLUOR™ 532-5-dUTP, ALEXA FLUOR™ 568-5-dUTP, ALEXA FLUOR™ 594-5-dUTP,ALEXA FLUOR™ 546-14-dUTP, fluorescein-12-UTP,tetramethylrhodamine-6-UTP, TEXAS RED™-5-UTP, mCherry, CASCADEBLUE™-7-UTP, BODIPY™ FL-14-UTP, BODIPY TMR-14-UTP, BODIPY™ TR-14-UTP,RHODAMINE GREEN™-5-UTP, ALEXA FLUOR™ 488-5-UTP, LEXA FLUOR™ 546-14-UTP(Molecular Probes, Inc. Eugene, Oreg.) and the like. Alternatively, theabove fluorophores and those mentioned herein can be added duringoligonucleotide synthesis using for example phosphoroamidite or NHSchemistry. Protocols are known in the art for custom synthesis ofnucleotides having other fluorophores (See, Henegariu et al. (2000)Nature Biotechnol. 18:345). 2-Aminopurine is a fluorescent base that canbe incorporated directly in the oligonucleotide sequence during itssynthesis. Nucleic acid could also be stained, a priori, with anintercalating dye such as DAPI, YOYO-1, ethidium bromide, cyanine dyes(e.g., SYBR Green) and the like.

Other fluorophores available for post-synthetic attachment include, butare not limited to, ALEXA FLUOR™ 350, ALEXA FLUOR™ 405, ALEXA FLUOR™430, ALEXA FLUOR™ 532, ALEXA FLUOR™ 546, ALEXA FLUOR™ 568, ALEXA FLUOR™594, ALEXA FLUOR™ 647, BODIPY 493/503, BODIPY FL, BODIPY R6G, BODIPY530/550, BODIPY TMR, BODIPY 558/568, BODIPY 558/568, BODIPY 564/570,BODIPY 576/589, BODIPY 581/591, BODIPY TR, BODIPY 630/650, BODIPY650/665, Cascade Blue, Cascade Yellow, Dansyl, lissamine rhodamine B,Marina Blue, Oregon Green 488, Oregon Green 514, Pacific Blue, PacificOrange, rhodamine 6G, rhodamine green, rhodamine red, tetramethylrhodamine, Texas Red (available from Molecular Probes, Inc., Eugene,Oreg.), Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy7 (Amersham Biosciences,Piscataway, N.J.) and the like. FRET tandem fluorophores can also beused, including, but not limited to, PerCP-Cy5.5, PE-Cy5, PE-Cy5.5,PE-Cy7, PE-Texas Red, APC-Cy7, PE-Alexa dyes (610, 647, and 680),APC-Alexa dyes and the like.

Metallic silver or gold particles can be used to enhance signal fromfluorescently labeled nucleotide and/or oligonucleotide sequences(Lakowicz et al. (2003) BioTechniques 34:62).

Other suitable labels for an oligonucleotide sequence can includefluorescein (FAM, FITC), digoxigenin, dinitrophenol (DNP), dansyl,biotin, bromodeoxyuridine (BrdU), hexahistidine (6×His), phosphor-aminoacids (e.g., P-tyr, P-ser, P-thr) and the like. The followinghapten/antibody pairs can be used for detection, in which each of theantibodies is derivatized with a detectable label: biotin/a-biotin,digoxigenin/a-digoxigenin, dinitrophenol (DNP)/a-DNP,5-Carboxyfluorescein (FAM)/a-FAM.

Detectable labels described herein are spectrally resolvable.“Spectrally resolvable” in reference to a plurality of fluorescentlabels means that the fluorescent emission bands of the labels aresufficiently distinct, i.e., sufficiently non-overlapping, thatmolecular tags to which the respective labels are attached can bedistinguished on the basis of the fluorescent signal generated by therespective labels by standard photodetection systems, e.g., employing asystem of band pass filters and photomultiplier tubes, or the like, asexemplified by the systems described in U.S. Pat. Nos. 4,230,558;4,811,218; or the like, or in Wheeless et al., pgs. 21-76, in FlowCytometry: Instrumentation and Data Analysis (Academic Press, New York,1985). Spectrally resolvable organic dyes, such as fluorescein,rhodamine, and the like, means that wavelength emission maxima arespaced at least 20 nm apart, and in another aspect, at least 40 nmapart. For chelated lanthanide compounds, quantum dots, and the like,spectrally resolvable means that wavelength emission maxima are spacedat least 10 nm apart, or at least 15 nm apart.

A reporter probe can comprise one or more cleavable linkermodifications. The one or more cleavable linker modifications can bepositioned anywhere in the reporter probe. A cleavable linkermodification can be located between the first and second domains of aprimary nucleic acid molecule of a reporter probe. FIG. 9 depicts anexemplary reporter probe of the present disclosure comprising acleavable linker modification between the first and second domains ofthe primary nucleic acid molecule. A cleavable linker modification canbe present between the first and second domains of the secondary nucleicacid molecules of a reporter probe. A cleavable linker modification canbe present between the first and second domains of the primary nucleicacid molecule and secondary nucleic acid molecules of a reporter probe.The left panel of FIG. 10 depicts an exemplary reporter probe of thepresent disclosure comprising cleavable linker modification between thefirst and second domains of the primary nucleic acid and between thefirst and second domains of the secondary nucleic acids.

A cleavable linker modification can be a compound of the Formula (I):

or a stereoisomer or salt thereof, wherein: R₁ is hydrogen, halogen,C₁₋₆alkyl, C₂₋₆alkenyl, C₂₋₆alkynyl, wherein said C₁₋₆alkyl,C₂₋₆alkenyl, C₂₋₆alkynl are each independently optionally substitutedwith at least one substituent R₁₀; R₂ is O, NH, or N(C₁₋₆alkyl); R₃ iscycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each optionallysubstituted with at least one substituent R₁₀; each R₄ and R₇ areindependently C₁₋₆alkyl, C₂₋₆alkenyl, C₂₋₆alkynyl, wherein saidC₁₋₆alkyl, C₂₋₆alkenyl, C₂₋₆alkynl are each independently optionallysubstituted with at least one substituent R₁₀; R₈ and R₉ are eachindependently cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, eachoptionally substituted with at least one substituent R₁₀, R₆ is O, NH orN(C₁₋₆alkyl); R₈ is O, NH, or N(C₁₋₆alkyl); each R₁₀ is independentlyhydrogen, halogen, —C₁₋₆alkyl, —C₂₋₆alkenyl, —C₂₋₆alkynyl,haloC₁₋₆alkyl, haloC₂₋₆alkenyl, haloC₂₋₆alkynyl, cycloalkyl,heterocyclyl, aryl, heteroaryl, —CN, —NO₂, oxo, —OR₁₁, —SO₂R₁₁, —SO₃ ⁻,—COR₁₁, —CO₂R₁₁, —CONR₁₁R₁₂, —C(═NR₁₁)NR₁₂R₁₃, —NR₁₁R₁₂, —NR₁₂COR₁₂,—NR₁₁CONR₁₂R₁₃, —NR₁₁CO₂R₁₂, —NR₁₁SONR₁₂R₁₃, —NR₁₁SO₂NR₁₂R₁₃, or—NR₁₁SO₂R₁₂; and R₁₁, R₁₂, and R₁₃, which may be the same or different,are each independently hydrogen, —C₁₋₆alkyl, —C₂₋₆alkenyl, —C₂₋₆alkynyl,haloC₁₋₆alkyl, haloC₂-6alkenyl, haloC₂₋₆alkynyl, C₁₋₆alkyloxyC₁₋₆alkyl-,cycloalkyl, heterocyclyl, aryl, or heteroaryl.

In one aspect, R₁ is C₁₋₆alkyl, preferably C₁₋₃alkyl such as methyl,ethyl, propyl or isopropyl; R₂ is NH or N(C₁₋₆alkyl); R₃ is a 5- to6-membered cycloalkyl, preferably cyclohexyl; R₄ is C₁₋₆alkyl,preferably C₁₋₃alkylene such as methylene, ethylene, propylene, orisopropylene; R₅ is a 5- to 6-membered heterocyclyl comprising onenitrogen atom and 0 or 1 additional heteroatoms selected from N, O andS, wherein said heterocyclyl is optionally substituted with one or twoR₁₀; R₆ is 0; R₇ is C₁₋₆alkyl, preferably C₁₋₃alkylene such asmethylene, ethylene, propylene, or isopropylene; R₈ is 0; R₉ is a 5- to6-membered heterocyclyl comprising one nitrogen atom and 0 or 1additional heteroatoms selected from N, O and S, wherein saidheterocyclyl is optionally substituted with one or two R₁₀; and each R₁₀is independently halogen, C₁₋₆alkyl, haloC₁₋₆alkyl, oxo, —SO₂H, or —SO₃⁻.

In one aspect, R₃ is cyclohexyl, R₄ is methylene, R₅ is1H-pyrrole-2,5-dione, and R₉ is pyrrolidine-2,5-dione, optionallysubstituted with SO₃ ⁻.

The linker compound can be

or a stereoisomer or salt thereof

The linker compound can be

or a stereoisomer or salt thereof.

The linker compound or linker modification can be

The linker compound or linker modification can be

Reporter probes can be assembled by mixing together three stocksolutions together with water. One stock solution contains primarynucleic acid molecules, one stock solution contains secondary nucleicacid molecules and the final stock solution contains the tertiarynucleic acid molecules. Table 2 depicts exemplary amounts of each stocksolution that can be mixed to assemble particular reporter probedesigns.

TABLE 2 Volume (μl) of Volume (μl) of secondary Volume (μl) of Reporterprimary nucleic nucleic acid tertiary nucleic Volume probe acidmolecules molecules (10 μM acid molecules (μl) of Design (10 μM stock)stock) (10 μM stock) Water 5 × 4 1 4.5 2.25 92.25 5 × 3 1 4.5 1.8 92.7 4× 4 1.28 4.5 2.25 91.97 4 × 3 1.28 4.5 1.8 92.42 3 × 4 1.8 4.5 2.2591.45

Target Nucleic Acid

The present disclosure provides methods for sequencing a nucleic acidusing the sequencing probes disclosed herein. The nucleic acid that isto be sequenced using the method of the present disclosure is hereinreferred to as a “target nucleic acid”. The term “target nucleic acid”shall mean a nucleic acid molecule (DNA, RNA, or PNA) whose sequence isto be determined by the probes, methods, and apparatuses of thedisclosure. In general, the terms “target nucleic acid”, “target nucleicacid molecule,”, “target nucleic acid sequence,” “target nucleic acidfragment,” “target oligonucleotide” and “target polynucleotide” are usedinterchangeably and are intended to include, but not limited to, apolymeric form of nucleotides that can have various lengths, eitherdeoxyribonucleotides or ribonucleotides, or analogs thereof.Non-limiting examples of nucleic acids include a gene, a gene fragment,an exon, an intron, intergenic DNA (including, without limitation,heterochromatic DNA), messenger RNA (mRNA), transfer RNA, ribosomal RNA,ribozymes, small interfering RNA (siRNA), non-coding RNA (ncRNA), cDNA,recombinant polynucleotides, branched polynucleotides, plasmids,vectors, isolated DNA of a sequence, isolated RNA of a sequence, nucleicacid probes, and primers. Prior to sequencing using the methods of thepresent disclosure, the identity and/or sequence of the target nucleicis known. Alternatively, the identity and/or sequence is unknown. It isalso possible that a portion of the sequence of a target nucleic acid isknown prior to sequencing using the methods of the present disclosure.For example, the method can be directed at determining a point mutationin a known target nucleic acid molecule.

The present methods directly sequence a nucleic acid molecule obtainedfrom a sample, e.g., a sample from an organism, and, preferably, withouta conversion (or amplification) step. As an example, for directRNA-based sequencing, the present methods do not require conversion ofan RNA molecule to a DNA molecule (i.e., via synthesis of cDNA) before asequence can be obtained. Since no amplification or conversion isrequired, a nucleic acid sequenced in the present disclosure will retainany unique base and/or epigenetic marker present in the nucleic acidwhen the nucleic acid is in the sample or when it was obtained from thesample. Such unique bases and/or epigenetic markers are lost insequencing methods known in the art.

The present methods can be used to sequence at single moleculeresolution. In other words, the present methods allow the user togenerate a final sequence based on data collected from a single targetnucleic acid molecule, rather than having to combine data from differenttarget nucleic acid molecules, preserving any unique features of thatparticular target.

The target nucleic acid can be obtained from any sample or source ofnucleic acid, e.g., any cell, tissue, or organism, in vitro, chemicalsynthesizer, and so forth. The target nucleic acid can be obtained byany art-recognized method. The nucleic acid can be obtained from a bloodsample of a clinical subject. The nucleic acid can be extracted,isolated, or purified from the source or samples using methods and kitswell known in the art.

A target nucleic acid can be fragmented by any means known in the art.Preferably, the fragmenting is performed by an enzymatic or a mechanicalmeans. The mechanical means can be sonication or physical shearing. Theenzymatic means can be performed by digestion with nucleases (e.g.,Deoxyribonuclease I (DNase I)) or one or more restriction endonucleases.

When a nucleic acid molecule comprising the target nucleic acid is anintact chromosome, steps should be taken to avoid fragmenting thechromosome.

The target nucleic acid can include natural or non-natural nucleotides,comprising modified nucleotides or nucleic acid analogues, as well-knownin the art.

The target nucleic acid molecule can include DNA, RNA, and PNA moleculesup to hundreds of kilobases in length (e.g. 1, 2, 3, 4, 5, 10, 20, 30,40, 50, 100, 200, 500, or more kilobases).

Capture Probes

The target nucleic acid can be immobilized (e.g., at one, two, three,four, five, six, seven, eight, nine, ten, or more positions) to asubstrate.

Exemplary useful substrates include those that comprise a binding moietyselected from the group consisting of ligands, antigens, carbohydrates,nucleic acids, receptors, lectins, and antibodies. The capture probecomprises a substrate binding moiety capable of binding with the bindingmoiety of the substrate. Exemplary useful substrates comprising reactivemoieties include, but are not limited to, surfaces comprising epoxy,aldehyde, gold, hydrazide, sulfhydryl, NETS-ester, amine, alkyne, azide,thiol, carboxylate, maleimide, hydroxymethyl phosphine, imidoester,isocyanate, hydroxyl, pentafluorophenyl-ester, psoralen, pyridyldisulfide or vinyl sulfone, polyethylene glycol (PEG), hydrogel, ormixtures thereof. Such surfaces can be obtained from commercial sourcesor prepared according to standard techniques. Exemplary usefulsubstrates comprising reactive moieties include, but are not limited to,OptArray-DNA NETS group (Accler8), Nexterion Slide AL (Schott) andNexterion Slide E (Schott).

The substrate can be any solid support known in the art, e.g., a coatedslide and a microfluidic device, which is capable of immobilizing atarget nucleic acid. The substrate can be a surface, membrane, bead,porous material, electrode or array. The substrate can be a polymericmaterial, a metal, silicon, glass or quartz for example. The targetnucleic acid can be immobilized onto any substrate apparent to those ofskill in the art.

When the substrate is an array, the substrate can comprise wells, thesize and spacing of which is varied depending on the target nucleic acidmolecule to be attached. In one example, the substrate is constructed sothat an ultra-dense ordered array of target nucleic acids is attached.Examples of the density of the array of target nucleic acids on asubstrate include from 500,000 to 10,000,000 target nucleic acidmolecules per mm², from 1,000,000 to 4,000,000 target nucleic acidmolecules per mm² or from 850,000 to 3,500,000 target nucleic acidmolecules per mm².

The wells in the substrate are locations for attachment of a targetnucleic acid molecule. The surface of the wells can be functionalizedwith reactive moieties described above to attract and bind specificchemical groups existing on the on the target nucleic acid molecules orcapture probes bound to the target nucleic acid molecules to attract,immobilize and bind the target nucleic acid molecule. These functionalgroups are well known to be able to specifically attract and bindbiomolecules through various conjugation chemistries.

For single nucleic acid molecule sequencing on a substrate such as anarray, a universal capture probe or universal sequence complementary tothe substrate binding moiety of a capture probe is attached to eachwell. A single target nucleic acid molecule is then bound to theuniversal capture probe or universal sequence complementary to thesubstrate binding moiety of a capture probe bound to the capture probeand sequencing can commence.

The target nucleic acid can be bound by one or more capture probes (i.e.two, three, four, five, six, seven, eight, nine, ten or more captureprobes). A capture probe comprises a domain that is complementary to aportion of the target nucleic acid and a domain that comprises asubstrate binding moiety. The portion of the target nucleic acid towhich a capture probe is complementary can be an end of the targetnucleic acid or not towards an end.

The substrate binding moiety of the capture probe can be biotin and thesubstrate can be avidin (e.g., streptavidin). Useful substratescomprising avidin are commercially available including TB0200 (Accelr8),SAD6, SAD20, SAD100, SAD500, SAD2000 (Xantec), SuperAvidin (Array-It),streptavidin slide (catalog #MPC 000, Xenopore) and STREPTAVIDINnslide(catalog #439003, Greiner Bio-one). The substrate binding moiety of thecapture probe can be avidin (e.g., streptavidin) and the substrate canbe biotin. Useful substrates comprising biotin that are commerciallyavailable include, but are not limited to, Optiarray-biotin (Accler8),BD6, BD20, BD100, BD500 and BD2000 (Xantec).

The substrate binding moiety of the capture probe can be a reactivemoiety that is capable of being bound to the substrate byphotoactivation. The substrate can comprise the photoreactive moiety, orthe first portion of the nanoreporter can comprise the photoreactivemoiety. Some examples of photoreactive moieties include aryl azides,such as N((2-pyridyldithio)ethyl)-4-azidosalicylamide; fluorinated arylazides, such as 4-azido-2,3,5,6-tetrafluorobenzoic acid;benzophenone-based reagents, such as the succinimidyl ester of4-benzoylbenzoic acid; and 5-Bromo-deoxyuridine.

The substrate binding moiety of a capture probe can be a nucleic acidthat can hybridize to a binding moiety of a substrate that iscomplementary. Each of the nucleic acids comprising a substrate bindingmoiety of a capture probe can independently be a canonical base or amodified nucleotide or nucleic acid analogue. At least one, at leasttwo, at least three, at least four, at least five, or at least sixnucleotides in the substrate binding moiety of a capture probe can bemodified nucleotides or nucleotide analogues. Typical ratios of modifiednucleotides or nucleotide analogues to canonical bases in a substratebinding moiety of a capture probe are 1:2 to 1:8. Typical modifiednucleotides or nucleic acid analogues useful in a substrate bindingmoiety of a capture probe are isoguanine and isocytosine.

The substrate binding moiety of the capture probe can be immobilized tothe substrate via other binding pairs apparent to those of skill in theart. After binding to the substrate, the target nucleic acid can beelongated by applying a force (e.g., gravity, hydrodynamic force,electromagnetic force “electrostretching”, flow-stretching, a recedingmeniscus technique, and combinations thereof) sufficient to extend thetarget nucleic acid. A capture probe can comprise or be associated witha detectable label, i.e., a fiducial spot.

The target nucleic acid can be bound by a second capture probe whichcomprises a domain that is complementary to a second portion of thetarget nucleic acid. The second portion of the target nucleic acid boundby the second capture probe is different than the first portion of thetarget nucleic acid bound by the first capture probe. The portion can bean end of the target nucleic acid or not towards an end. Binding of asecond capture probe can occur after or during elongation of the targetnucleic acid or to a target nucleic acid that has not been elongated.The second capture probe can have a binding as described above.

The target nucleic acid can be bound by a third, fourth, fifth, sixth,seventh, eighth, ninth or tenth capture probe which comprises a domainthat is complementary to a third, fourth, fifth, sixth, seventh, eighth,ninth or tenth portion of the target nucleic acid. The portion can be anend of the target nucleic acid or not towards an end. Binding of athird, fourth, fifth, sixth, seventh, eighth, ninth or tenth captureprobe can occur after or during elongation of the target nucleic acid orto a target nucleic acid that has not been elongated. The third, fourth,fifth, sixth, seventh, eighth, ninth or tenth capture probe can have abinding as described above.

The capture probe is capable of isolating a target nucleic acid from asample. Here, a capture probe is added to a sample comprising the targetnucleic acid. The capture probe binds the target nucleic acid via theregion of the capture probe that his complementary to a region of thetarget nucleic acid. When the target nucleic acid contacts a substratecomprising a moiety that binds the capture probe's substrate bindingmoiety, the nucleic acid becomes immobilized onto the substrate.

FIG. 11 shows the capture of a target nucleic acid using a two captureprobe system of the present disclosure. Genomic DNA is denatured at 95°C. and hybridized to a pool of capture reagents. This pool of capturereagents comprise the oligonucleotides Probe A, Probe B, and anti-senseblock probes. Probe A comprises a biotin moiety at the 3′ end of theprobe and a sequence that is complementary to the 5′ end of the targetnucleic acid. Probe B comprises a purification binding sequence that canbe bound by paramagnetic beads at the 5′ end of the probe and anucleotide sequence that is complementary to the 3′ end of the targetnucleic acid. The anti-sense block probe comprises a nucleotide sequencethat is complementary to the anti-sense strand of the portion of thetarget nucleic acid that is to be sequenced. After hybridization withthe capture reagents, a sequencing window is created on the targetnucleic acid between the hybridized Probe A and Probe B. The targetnucleic acid is purified using paramagnetic beads that bind to the 5′sequence of Probe B. Any excess capture reagents or complementaryanti-sense DNA strands are washed away, resulting in the purification ofthe intended target nucleic acid. The purified target nucleic acid isthen flowed through a flow chamber that includes a surface that can bindto the biotin moiety on the hybridized Probe A, such as streptavidin.This results in the tethering of one end of the target nucleic acid tothe surface of the flow cell. To capture the other end, the targetnucleic acid is flow-stretched and a biotinylated probe complementary tothe purification binding sequence of Probe B is added. Upon hybridizingto the purification binding sequence of Probe B, the biotinylated probecan bind to the surface of the flow cell, resulting in a captured targetnucleic acid molecule that is elongated and bound to the flow cellsurface at both ends.

To ensure that a user “captures” as many target nucleic acid moleculesas possible from high fragmented samples, it is helpful to include aplurality of capture probes, each complementary to a different region ofthe target nucleic acid. For example, there can be three pools ofcapture probes, with a first pool complementary to regions of the targetnucleic acid near its 5′ end, a second pool complementary to regions inthe middle of the target nucleic acid, and a third pool near its 3′ end.This can be generalized to “n-regions-of-interest” per target nucleicacid. In this example, each individual pool of fragmented target nucleicacid bound to a capture probe comprising or bound to a biotin tag. 1/nthof input sample (where n=the number of distinct regions in targetnucleic acid) is isolated for each pool chamber. The capture probe bindsthe target nucleic acid of interest. Then the target nucleic acid isimmobilized, via the capture probe's biotin, to an avidin moleculeadhered to the substrate. Optionally, the target nucleic acid isstretched, e.g., via flow or electrostatic force. All n-pools can bestretched-and-bound simultaneously, or, in order to maximize the numberof fully stretched molecules, pool 1 (which captures most 5′ region) canbe stretched and bound first; then pool 2, (which captures themiddle-of-target region) is then can be stretched and bound; finally,pool 3 is can be stretched and bound.

The present disclosure also allows a user to capture and concurrentlysequence a plurality of target nucleic acids, a plurality of captureprobes can be hybridized to a mixed sample of target nucleic acids. Aplurality of target nucleic acids can include a group of more than onenucleic acid, in which each nucleic acid contains the same sequence, ora group of more than one nucleic acid, in which each nucleic acid doesnot necessarily contain the same sequence. Likewise, the plurality ofcapture probes can include either a group of more than one capture probethat are identical in sequence, or a group of more than one captureprobe that are not necessarily identical in sequence. For example, usinga plurality of capture probes that all contain the same sequence canallow the user to capture a plurality of target nucleic acids that allcontain the same sequence. By sequencing this plurality of targetnucleic acids containing the same sequence, a higher level of sequencingaccuracy can be achieved due to data redundancy. In another example, twoor more specific genes of interest can be captured and sequencedconcurrently using a group of capture probes that includes captureprobes complementary to each gene of interest. This allows the user toperform multiplexed sequencing of specific genes. FIG. 12 shows theresults from an experiment using the present methods to capture anddetect a multiplex cancer panel, composed of 100 targets, using a FFPEsample.

When complete sequencing coverage is desired, the number of distinctcapture probes required is inversely related to the size of targetnucleic acid fragment. In other word, more capture probes will berequired for a highly-fragmented target nucleic acid. For sample typeswith highly fragmented and degraded target nucleic acids (e.g.,Formalin-Fixed Paraffin Embedded Tissue) it can be useful to includemultiple pools of capture probes. On the other hand, for samples withlong target nucleic acid fragments, e.g., in vitro obtained isolatednucleic acids, a single capture probe at a 5′ end can be sufficient.

The region of the target nucleic acid between two capture probes orafter one capture probe and before a terminus of the target nucleic acidis referred herein as a “sequencing window”. The sequencing windowcreated when two capture probes are used to capture a target nucleicacid is labeled in FIG. 11. The sequencing window is a portion of thetarget nucleic acid that is available to be bound by a sequencing probe.The minimum sequencing window is a target binding domain length (e.g., 4to 10 nucleotides) and a maximum sequencing window is the majority of awhole chromosome.

When large target nucleic acid molecules are sequenced using the presentmethods, a “blocker oligo” or a plurality of blocker oligos can behybridized along the length of the target nucleic acid to control thesize of the sequencing window. Blocker oligos hybridize to the targetnucleic acid at specific locations, thereby preventing the binding ofsequencing probes at those locations, creating smaller sequencingwindows of interest. FIG. 13 shows a schematic of two captured targetDNA molecules hybridized to capture probes, blocker oligos, andsequencing probes. By creating smaller sequencing windows, thesequencing reactions is confined to specific regions of interest on thetarget DNA molecule, increasing the speed and accuracy of sequencing.The use of blocker oligos is particularly useful when sequencingparticular mutations at known locations within a target nucleic acid, asthe entire target nucleic acid does not need to be sequenced. Theexample in FIG. 13 shows the targeted sequencing of two heterozygoussites to distinguish between two different haplotypes.

Methods of the Present Disclosure

The sequencing method of the present disclosure comprises reversiblyhybridizing at least one sequencing probe disclosed herein to a targetnucleic acid.

A method for sequencing a nucleic acid can comprise (1) hybridizing asequencing probe described herein to a target nucleic acid. The targetnucleic acid can optionally be immobilized to a substrate at one or morepositions. An exemplary sequencing probe can comprise a target bindingdomain and a barcode domain; wherein the target binding domain comprisesat least eight nucleotides hybridized to the target nucleic acid,wherein at least six nucleotides in the target binding domain canidentify a corresponding nucleotide in the target nucleic acid molecule(for example, when the target binding domain sequence is exactly sixnucleotides, those six nucleotides identify the complementary sixnucleotides with the target molecule to which it is hybridized) andwherein at least two nucleotides in the target binding domain do notidentify a corresponding nucleotide in the target nucleic acid molecule(for example, those at least two nucleotides do not identify thecomplementary two nucleotides with the target molecule to which it ishybridized); wherein at least two nucleotides of the at least sixnucleotides in the target binding domain are modified nucleotides ornucleotide analogues; wherein the barcode domain comprises a syntheticbackbone, the barcode domain comprising at least three attachmentpositions, each attachment position comprising at least one attachmentregion comprising at least one nucleic acid sequence capable of beingbound by a complementary nucleic acid molecule, wherein each attachmentposition of the at least three attachment positions corresponds to twonucleotides of the at least six nucleotides in the target binding domainand each of the at least three attachment positions have a differentnucleic acid sequence, and wherein the nucleic acid sequence of eachposition of the at least three attachment positions determines theposition and identity of the corresponding two nucleotides of the atleast six nucleotides in the target nucleic acid that is bound by thetarget binding domain.

Following hybridizing of a sequencing probe to the target nucleic acid,the method comprises (2) binding a first complementary nucleic acidmolecule comprising a first detectable label and an at least seconddetectable label to a first attachment position of the at least threeattachment positions of the barcode domain; (3) detecting the first andat least second detectable label of the bound first complementarynucleic acid molecule; (4) identifying the position and identity of atleast two nucleotides in the immobilized target nucleic acid. Forexample, when the first complementary nucleic acid molecule comprisestwo detectable labels, the two detectable labels identify the at leasttwo nucleotides in the immobilized target nucleic acid.

Following detection of the at least two detectable labels, removing theat least two detectable labels from the first complementary nucleic acidmolecule. Thus, the method further comprises (5) binding to the firstattachment position a first hybridizing nucleic acid molecule lacking adetectable label, thereby unbinding the first complementary nucleic acidmolecule comprising the detectable labels, or contacting the firstcomplementary nucleic acid molecule comprising the detectable labelswith a force sufficient to release the first detectable label and atleast second detectable label. Thus, following step (5) no detectablelabels are bound to the first attachment positions. The method furthercomprises (6) binding a second complementary nucleic acid moleculecomprising a third detectable label and an at least fourth detectable toa second attachment position of the at least three attachment positionsof the barcode domain; (7) detecting the third and at least fourthdetectable label of the bound second complementary nucleic acidmolecule; (8) identifying the position and identity of at least twonucleotides in the optionally immobilized target nucleic acid; (9)repeating steps (5) to (8) until each attachment position of the atleast three attachment positions in the barcode domain have been boundby a complementary nucleic acid molecule comprising two detectablelabels, and the two detectable labels of the bound complementary nucleicacid molecule has been detected, thereby identifying the linear order ofat least six nucleotides for at least a first region of the immobilizedtarget nucleic acid that was hybridized by the target binding domain ofthe sequencing probe; and (10) removing the sequencing probe from theoptionally immobilized target nucleic acid.

The method can further comprise (11) hybridizing a second sequencingprobe to a target nucleic acid that is optionally immobilized to asubstrate at one or more positions, and wherein the target bindingdomain of the first sequencing probe and the second sequencing probe aredifferent; (12) binding a first complementary nucleic acid moleculecomprising a first detectable label and an at least second detectablelabel to a first attachment position of the at least three attachmentpositions of the barcode domain; (13) detecting the first and at leastsecond detectable label of the bound first complementary nucleic acidmolecule; (14) identifying the position and identity of at least twonucleotides in the optionally immobilized target nucleic acid; —(15)binding to the first attachment position a first hybridizing nucleicacid molecule lacking a detectable label, thereby unbinding the firstcomplementary nucleic acid molecule or complex comprising the detectablelabels, or contacting the first complementary nucleic acid molecule orcomplex comprising the detectable labels with a force sufficient torelease the first detectable label and at least second detectable label;(16) binding a second complementary nucleic acid molecule comprising athird detectable label and an at least fourth detectable label to asecond attachment position of the at least three attachment positions ofthe barcode domain; (17) detecting the third and at least fourthdetectable label of the bound second complementary nucleic acidmolecule; (18) identifying the position and identity of at least twonucleotides in the immobilized target nucleic acid; (19) repeating steps(15) to (18) until each attachment position of the at least threeattachment positions in the barcode domain have been bound by acomplementary nucleic acid molecule comprising two detectable labels,and the two detectable labels of the bound complementary nucleic acidmolecule has been detected, thereby identifying the linear order of atleast six nucleotides for at least a second region of the immobilizedtarget nucleic acid that was hybridized by the target binding domain ofthe sequencing probe; and (20) removing the second sequencing probe fromthe optionally immobilized target nucleic acid.

The method can further comprise assembling each identified linear orderof nucleotides in the at least first region and at least second regionof the immobilized target nucleic acid, thereby identifying a sequencefor the immobilized target nucleic acid.

Steps (5) and (6) can occur sequentially or concurrently. The first andat least second detectable labels can have the same emission spectrum orcan have different emission spectra. The third and at least fourthdetectable labels can have the same emission spectrum or can havedifferent emission spectra.

The first complementary nucleic acid molecule can comprise a cleavablelinker. The second complementary nucleic acid molecule can comprise acleavable linker. The first complementary nucleic acid molecule and thesecond complementary nucleic acid molecule can each comprise a cleavablelinker. Preferably, the cleavable linker is photo-cleavable. The releaseforce can be light. Preferably, UV light. The light can be provided by alight source selected from the group consisting of an arc-lamp, a laser,a focused UV light source, and light emitting diode.

The first complementary nucleic acid molecule and the first hybridizingnucleic acid molecule lacking a detectable label can comprise the samenucleic acid sequence. For example, the first hybridizing nucleic acidmolecule lacking a detectable label can comprise the same nucleic acidsequence as that portion of the first complementary nucleic acidmolecule that binds to a first attachment position of the at least threeattachment positions of the barcode domain. The first hybridizingnucleic acid molecule lacking a detectable label can comprise a nucleicacid sequence complementary to a flanking single-stranded polynucleotideadjacent to the first attachment position in the barcode domain.

The second complementary nucleic acid molecule and the secondhybridizing nucleic acid molecule lacking a detectable label cancomprise the same nucleic acid sequence. The second hybridizing nucleicacid molecule lacking a detectable label can comprise a nucleic acidsequence complementary to a flanking single-stranded polynucleotideadjacent to the second attachment position in the barcode domain.

The present invention further provides methods of sequencing a nucleicacid utilizing a plurality of sequencing probes disclosed herein. Forexample, the target nucleic acid is hybridized to more than onesequencing probe and each probe can sequence the portion of the targetnucleic acid to which it is hybridized.

The present disclosure also provides a method for sequencing a nucleicacid comprising (1) hybridizing at least one first population of firstsequencing probes comprising a plurality of the sequencing probesdescribed herein to a target nucleic acid that is optionally immobilizedto a substrate at one or more positions; (2) binding a firstcomplementary nucleic acid molecule comprising a first detectable labeland an at least second detectable label to a first attachment positionof the at least three attachment positions of the barcode domain; (3)detecting the first and at least second detectable label of the boundfirst complementary nucleic acid molecule; (4) identifying the positionand identity of at least two nucleotides in the immobilized targetnucleic acid; (5) binding to the first attachment position a firsthybridizing nucleic acid molecule lacking a detectable label, therebyunbinding the first complementary nucleic acid molecule comprising thedetectable labels, or contacting the first complementary nucleic acidmolecule comprising the detectable labels with a force sufficient torelease the first detectable label and at least second detectable label;(6) binding a second complementary nucleic acid molecule comprising athird detectable label and an at least fourth detectable to a secondattachment position of the at least three attachment positions of thebarcode domain; (7) detecting the third and at least fourth detectablelabel of the bound second complementary nucleic acid molecule; (8)identifying the position and identity of at least two nucleotides in theoptionally immobilized target nucleic acid; (9) repeating steps (5) to(8) until each attachment position of the at least three attachmentpositions in the barcode domain have been bound by a complementarynucleic acid molecule comprising two detectable labels, and the twodetectable labels of the bound complementary nucleic acid molecule hasbeen detected, thereby identifying the linear order of at least sixnucleotides for at least a first region of the immobilized targetnucleic acid that was hybridized by the target binding domain of thesequencing probe; and (10) removing the at least one first population offirst sequencing probes from the optionally immobilized target nucleicacid.

The method can further comprise (11) hybridizing at least one secondpopulation of second sequencing probes comprising a plurality of thesequencing probes disclosed herein to a target nucleic acid that isoptionally immobilized to a substrate at one or more positions, andwherein the target binding domain of the first sequencing probe and thesecond sequencing probe are different; (12) binding a firstcomplementary nucleic acid molecule comprising a first detectable labeland an at least second detectable label to a first attachment positionof the at least three attachment positions of the barcode domain; (13)detecting the first and at least second detectable label of the boundfirst complementary nucleic acid molecule; (14) identifying the positionand identity of at least two nucleotides in the optionally immobilizedtarget nucleic acid; (15) binding to the first attachment position afirst hybridizing nucleic acid molecule lacking a detectable label,thereby unbinding the first complementary nucleic acid molecule orcomplex comprising the detectable labels, or contacting the firstcomplementary nucleic acid molecule or complex comprising the detectablelabels with a force sufficient to release the first detectable label andat least second detectable label; (16) binding a second complementarynucleic acid molecule comprising a third detectable label and an atleast fourth detectable label to a second attachment position of the atleast three attachment positions of the barcode domain; (17) detectingthe third and at least fourth detectable label of the bound secondcomplementary nucleic acid molecule; (18) identifying the position andidentity of at least two nucleotides in the immobilized target nucleicacid; (19) repeating steps (15) to (18) until each attachment positionof the at least three attachment positions in the barcode domain havebeen bound by a complementary nucleic acid molecule comprising twodetectable labels, and the two detectable labels of the boundcomplementary nucleic acid molecule has been detected, therebyidentifying the linear order of at least six nucleotides for at least asecond region of the immobilized target nucleic acid that was hybridizedby the target binding domain of the sequencing probe; and (20) removingthe at least one second population of second sequencing probes from theoptionally immobilized target nucleic acid.

The method can further comprise assembling each identified linear orderof nucleotides in the at least first region and at least second regionof the immobilized target nucleic acid, thereby identifying a sequencefor the immobilized target nucleic acid.

Steps (5) and (6) can occur sequentially or concurrently. The first andat least second detectable labels can have the same emission spectrum orcan have different emission spectra. The third and at least fourthdetectable labels can have the same emission spectrum or can havedifferent emission spectra.

The first complementary nucleic acid molecule can comprise a cleavablelinker. The second complementary nucleic acid molecule can comprise acleavable linker. The first complementary nucleic acid molecule and thesecond complementary nucleic acid molecule can each comprise a cleavablelinker. Preferably, the cleavable linker is photo-cleavable. The releaseforce can be light. Preferably, UV light. The light can be provided by alight source selected from the group consisting of an arc-lamp, a laser,a focused UV light source, and light emitting diode.

The first complementary nucleic acid molecule and the first hybridizingnucleic acid molecule lacking a detectable label can comprise the samenucleic acid sequence. The first hybridizing nucleic acid moleculelacking a detectable label can comprise a nucleic acid sequencecomplementary to a flanking single-stranded polynucleotide adjacent tothe first attachment position in the barcode domain.

The second complementary nucleic acid molecule and the secondhybridizing nucleic acid molecule lacking a detectable label cancomprise the same nucleic acid sequence. The second hybridizing nucleicacid molecule lacking a detectable label can comprise a nucleic acidsequence complementary to a flanking single-stranded polynucleotideadjacent to the second attachment position in the barcode domain.

The sequencing methods are further described herein.

FIG. 14 shows a schematic overview of a single exemplary sequencingcycle of the present disclosure. Although immobilizing a target nucleicacid prior to sequencing is not required for the instant methods, inthis example, the method begins with a target nucleic acid that has beencaptured using capture probes and bound to a flow cell surface as shownin the left upper-most panel. A pool of sequencing probes is then flowedinto the flow cell to allow sequencing probes to hybridize to the targetnucleic acid. In this example, the sequencing probes are those depictedin FIG. 1. These sequencing probes comprise a 6-mer sequence within thetarget binding domain that hybridizes to the target nucleic acid. The6-mer is flanked on either side by (N) bases which can be auniversal/degenerate base or composed of any of the four canonical basesthat is not specific to the target dictated by bases b₁-b₂-b₃-b₄-b₅-b₆.Using 6-mer sequences, a set of 4096 (4{circumflex over ( )}6)sequencing probes enables the sequencing of any target nucleic acid. Forthis example, the set of 4096 sequencing probes are hybridized to thetarget nucleic acid in 8 pools of 512 sequencing probes each. The 6-mersequences in the target binding domain of the sequencing probes willhybridize along the length of the target nucleic acid at positions wherethere is a perfect complementary match between the 6-mer and the targetnucleic acid, as shown in upper middle panel of FIG. 14. In thisexample, a single sequencing probe hybridizes to the target nucleicacid. Any unbound sequencing probes are washed out of the flow cell.

These sequencing probes also comprise a barcode domain with threeattachment positions R₁, R₂ and R₃, as described above. The attachmentregions within attachment position R₁ comprise one or more nucleotidesequences that correspond to the first dinucleotide of the 6-mer of thesequencing probe. Thus, only reporter probes comprising complementarynucleic acids that correspond to the identity of the first dinucleotidepresent in the target binding domain of the sequencing probe willhybridize to attachment position R₁. Likewise, the attachment regionswithin attachment position R₂ of the sequencing probe correspond to thesecond dinucleotide present in the target binding domain and theattachment regions within attachment position R₃ of the sequencing probecorrespond to the second dinucleotide present in the target bindingdomain

The method continues in the right upper-most panel of FIG. 14. A pool ofreporter probes is flowed into the flow cell. Each reporter probe in thereporter probe pool comprises a detectable label, in the form of a dualcolor combination, and a complementary nucleic acid that can hybridizeto a corresponding attachment region within the attachment position R₁of a sequencing probe. The dual color combination and the complementarynucleic acid of a particular reporter probe correspond to one of 16possible dinucleotides, as described above. Each pool of reporter probesis designed such that the dual color combination that corresponds to aspecific dinucleotide is established before sequencing. For example, inthe sequencing experiment depicted in FIG. 14, for the first pool ofreporter probes that is hybridized to attachment position R₁, the dualcolor combination Yellow-Red can correspond to the dinucleotideAdenine-Thymine. After hybridization of the reporter probe to attachmentposition R₁, as shown in the upper right panel of FIG. 14, any unboundreporter probes are then washed out of the flow cell and the detectablelabel of the bound reporter probe is recorded to determine the identityof the first dinucleotide of the 6-mer.

The detectable label attributed to the reporter probe hybridized toattachment position R₁ is removed. To remove the detectable label, thereporter probe can include a cleavable linker and the addition of theappropriate cleaving agent can be added. Alternatively, a complementarynucleic acid lacking a detectable label is hybridized to attachmentposition R₁ of the sequencing probe and displaces the reporter probewith the detectable label. Irrespective of the method of removing thedetectable label, the attachment position R₁ no longer emits adetectable signal. The process by which an attachment position of abarcode domain that was previously emitting a detectable signal isrendered no longer able to emit a detectable signal is referred toherein as “darkening”.

A second pool of reporter probes is flowed into the flow cell. Eachreporter probe in the reporter probe pool comprises a detectable label,in the form of a dual color combination, and a complementary nucleicacid that can hybridize to a corresponding attachment region withinattachment position R₂ of a sequencing probe. The dual color combinationand the complementary nucleic acid of a particular reporter probecorrespond to one of 16 possible dinucleotides. It is possible that aparticular dual color combination corresponds to one dinucleotide in thecontext of the first pool of reporter probes, and a differentdinucleotide in the context of the second pool of reporter probes. Afterhybridization of the reporter probes to attachment position R₂, as shownin the bottom right panel of FIG. 14, any unbound reporter probes arethen washed out of the flow cell and the detectable label is recorded todetermine the identity of the second dinucleotide of the 6-mer presentin the sequencing probe.

To remove the detectable label at position R₂, the reporter probe caninclude a cleavable linker and the addition of the appropriate cleavingagent can be added. Alternatively, a complementary nucleic acid lackinga detectable label is hybridized to attachment position R₂ of thesequencing probe and displaces the reporter probe with the detectablelabel. Irrespective of the method of removing the detectable label, theattachment position R₂ no longer emits a detectable signal.

A third pool of reporter probes is then flowed into the flow cell. Eachreporter probe in the third reporter probe pool comprises a detectablelabel, in the form of a dual color combination, and a complementarynucleic acid that can hybridize to a corresponding attachment regionwithin attachment position R₃ of a reporter probe. The dual colorcombination and the complementary nucleic acid of a particular reporterprobe correspond to one of 16 possible dinucleotides. Afterhybridization of the reporter probes to position R₃, as shown in thebottom middle panel of FIG. 14, any unbound reporter probes are thenwashed out of the flow cell and the detectable label is recorded todetermine the identity of the third dinucleotide of the 6-mer present inthe sequencing probe. In this way, all three dinucleotides of the targetbinding domain are identified and can be assembled together to revealthe sequence of the target binding domain and therefore the sequence ofthe target nucleic acid.

To continue to sequence the target nucleic acid, any bound sequencingprobes can be removed from the target nucleic acid. The sequencing probecan be removed from the target nucleic acid even if a reporter probe isstill hybridized to position R₃ of the barcode domain. Alternatively,the reporter probe hybridized to position R₃ can be removed from thebarcode domain prior to the removal of the sequencing probe from thetarget binding domain, for example, by using the darkening procedures asdescribed above for reporters at positions R₁ and R₂.

The sequencing cycle depicted in FIG. 14 can be repeated any number oftimes, beginning each sequencing cycle either with the hybridization ofthe same pool of sequencing probes to the target nucleic acid moleculeor with the hybridization of a different pool of sequencing probes tothe target nucleic acid. It is possible that the second pool ofsequencing probes bind to the target nucleic acid at a position thatoverlaps the position at which the first sequencing probe or pool ofsequencing probes were bound during the first sequencing cycle. Therebycertain nucleotides within the target nucleic acid can be sequenced morethan once and using more than one sequencing probe.

FIG. 15 depicts a schematic of one full cycle of the sequencing methodof the present disclosure and the corresponding imaging data collectedduring this cycle. In this example, the sequencing probe used are thosedepicted in FIG. 1 and the sequencing steps are the same as thosedepicted in FIG. 14 and described above. After the sequencing domain ofthe sequencing probe is hybridized to the target nucleic acid, areporter probe is hybridized to the first attachment position (R₁) ofthe sequencing probe. The first reporter probe is then imaged to recordcolor dots. In FIG. 15, the color dots are labeled with dotted circles.The color dots correspond to a single sequencing probe that is beingrecorded during the full cycle. In this example, 7 sequencing probes arerecorded (1 to 7). The first attachment position of the barcode domainis then darkened and a dual fluorescence reporter probe is hybridized tothe second attachment position (R₂) of the sequencing probe. The secondreporter probe is then imaged to record color dots. The secondattachment position of the barcode domain is then darkened and a dualfluorescence reporter is hybridized to the third attachment position(R₃) of the sequencing probe. The third reporter probe is then imaged torecord color dots. The three color dots from each sequencing probe 1 to7 are then arranged in order. Each color spot is then mapped to aspecific dinucleotide using the decoding matrix to reveal the sequenceof the target binding domain of sequencing probes 1 to 7.

During a single sequencing cycle, the number of reporter probe poolsneeded to determine the sequence of the target binding domain of anysequencing probes bound to a target nucleic acid is identical to thenumber of attachment positions in the barcode domain. Thus, for abarcode domain having three positions, three reporter probe pools willbe cycled over the sequencing probes.

A pool of sequencing probes can comprise a plurality of sequencingprobes that are all identical in sequence or a plurality of sequencingprobes that are not all identical in sequence. When a pool of sequencingprobes include a plurality of sequencing probes that are not allidentical in sequence, each different sequencing probe can be present inthe same number, or different sequencing probes can be present indifferent numbers.

FIG. 16 shows an exemplary sequencing probe pool configuration of thepresent disclosure in which the eight color combinations specified aboveare used to design eight different pools of sequencing probes when thesequencing probe contains: (a) a target binding domain that has 6nucleotides (6-mer) that specifically binds to the target nucleic acidand (b) three attachment positions (R₁, R₂ and R₃) in the barcodedomain. There are a possible 4096 unique 6-mer sequences(4×4×4×4×4×4=4096). Given that each of the three attachment positions inthe barcode domain can be hybridized to a complementary nucleic acidbound by one of eight different color combinations, there are 512 uniquesets of 3 color combinations possible (8*8*8=512). For example, a probewhere R₁ hybridizes to a complementary nucleic acid bound to the colorcombination GG, R₂ hybridizes to a complementary nucleic acid bound tothe color combination BG, and R₃ hybridizes to a complementary nucleicacid bound to the color combination YR, the set of 3 color combinationsis accordingly GG-BG-YR. Within a pool of sequencing probes, each uniqueset of three color combinations will correspond to a unique 6mer withinthe target binding domain. Given each pool contains 512 unique 6mers,and there are a total of 4096 possible 6mers, eight pools are needed tosequence all possible 6mers (4096/512=8). The specific sequencing probesthat are placed in each of the 8 pools is determined to ensure optimalhybridization of each sequencing probe to the target nucleic acid. Toensure optimal hybridization several precautions are taken including:(a) separating perfect 6mer complements into different pools; (b)separating 6mers with a high Tm and a low Tm into different pools; and(c) separating 6mers into different pools based on empirically-learnedhybridization patterns.

FIG. 17 shows the difference between the sequencing probes described inUS Patent Publication No. 20160194701 and the sequencing probes of thepresent disclosure. As depicted on the left panel of FIG. 17, US PatentPublication No. 20160194701 describes a sequencing probe with a barcodedomain that comprises six attachment positions that are hybridized tocomplementary nucleic acids. Each complementary nucleic acids is boundto one of four different fluorescent dyes. In this configuration, eachcolor (red, blue, green, yellow) corresponds to one nucleotide (A, T, C,or G) in the target binding domain. This probe design creates 4096unique probes (4{circumflex over ( )}6). As depicted in the right panelof FIG. 17, in one example of the present disclosure, the barcode domainof each sequencing probe comprises 3 attachment positions that arehybridized to complementary nucleic acids, as depicted in the rightpanel of FIG. 17. Unlike US Patent Publication No. 20160194701, thesecomplementary nucleic acids are bound by 1 of 8 different colorcombinations (GG, RR, GY, RY, YY, RG, BB, and RB). Each colorcombination corresponds to a specific dinucleotide in the target bindingdomain. This configuration creates 512 unique probes (8{circumflex over( )}3). To cover all possible hexamer combinations within a targetbinding domain (4096), 8 separate pools of these 512 unique probes areneeded to sequence an entire target nucleic acid. Since 8 colorcombinations are used to label the complementary nucleic acid, but thereare 16 possible dinucleotides, certain color combinations willcorrespond to different dinucleotides depending on which pool ofsequencing probes is being used. For example, in FIG. 17, in the 1^(st),2^(nd), 3^(rd), and 4^(th) pools of sequencing probes, the colorcombination BB corresponds to the dinucleotide AA and the colorcombination GG corresponds to the dinucleotide AT. In the 5^(th), 6^(th), 7^(th), and 8^(th) pools of sequencing probes, the colorcombination BB corresponds to the dinucleotide CA and the colorcombination CT corresponds to the dinucleotide AT.

A plurality of sequencing probes (i.e. more than one sequencing probe)can be hybridized within the sequencing window. During sequencing, theidentity and spatial position of the detectable labels bound to eachsequencing probe in the plurality of hybridized sequencing probes isrecorded. This allows for subsequent identification of both the positionand identity of a plurality of dinucleotides. In other words, byhybridizing a plurality of sequencing probes simultaneously to a singletarget nucleic acid molecule, multiple positions along the targetnucleic acid can be sequenced concurrently, increasing the speed ofsequencing.

FIG. 18 shows a schematic of a single sequencing probe and a pluralityof sequencing probes hybridized to a captured target nucleic acidmolecule. The sequencing window between the two hybridized 5′ and 3′capture probes allows for the hybridization of a single sequencing probe(left panel) or a plurality of sequencing probes (right panel) along thelength of the target nucleic acid molecule. By hybridizing a pluralityof sequencing probes along the length of the target nucleic acidmolecule, more than one location on the target nucleic acid molecule canbe sequence concurrently, increasing the speed of sequencing. FIG. 19shows fluorescence images recorded during the sequencing method of thepresent disclosure when a single probe (left panel) or a plurality ofprobes (right panel) are hybridized to a captured target nucleic acid.The right panel of FIG. 19 shows that the fluorescence signal fromindividual probes of a plurality of probes bound along the length of atarget nucleic acid can be spatially resolved.

FIG. 20 shows a schematic of a plurality of sequencing probes of thepresent disclosure bound along the length of a target nucleic acid thatis 15 kilobases in length and the corresponding recorded fluorescenceimages. Sequencing probes can bind at even intervals along the length oftarget nucleic acid, as shown in the right panel of FIG. 20. Sequencingprobes need not bind at even intervals along the length of a targetnucleic acid, as shown in the left panel of FIG. 20. The fluorescenceimages shown in FIG. 20 demonstrate the signals from a plurality ofsequencing probes bound along the length of a target nucleic acid can bespatially resolved to obtain sequencing information at multiplelocations of a target nucleic acid concurrently.

The distribution of probes along a length of target nucleic acid iscritical for resolution of detectable signal. There are occasions whentoo many probes in a region can cause overlap of their detectable label,thereby preventing resolution of two nearby probes. This is explained asfollows. Given that one nucleotide is 0.34 nm in length and given thatthe lateral (x-y) spatial resolution of a sequencing apparatus is about200 nm, a sequencing apparatus's resolution limit is about 588 base pair(i.e., a 1 nucleotide/0.34 nm×200 nm). That is to say, the sequencingapparatus mentioned above would be unable to resolve signals from twoprobes hybridized to a target nucleic acid when the two probes arewithin about 588 base pair of each other. Thus, two probes, depending onthe resolution of the sequencing apparatus, will need be spacedapproximately 600 bp's apart before their detectable label can beresolved as distinct “spots”. So, at optimal spacing, there should be asingle probe per 600 bp of target nucleic-acid. Preferably, eachsequencing probe in a population of probes will bind no closer than 600nucleotides from each other. A variety of software approaches (e.g.,utilize fluorescence intensity values and wavelength dependent ratios)can be used to monitor, limit, and potentially deconvolve the number ofprobes hybridizing inside a resolvable region of a target nucleic acidand to design probe populations accordingly. Moreover, detectable labels(e.g., fluorescent labels) can be selected that provide more discretesignals. Furthermore, methods in the literature (e.g., Small andParthasarthy: “Superresolution localization methods.” Annu. Rev. PhysChem., 2014; 65:107-25) describe structured-illumination and a varietyof super-resolution approaches which decrease the resolution limit of asequencing microscope up to 10's-of-nanometers. Use of higher resolutionsequencing apparatuses allow for use of probes with shorter targetbinding domains.

As mentioned above, designing the Tm of probes can affect the number ofprobes hybridized to a target nucleic acid. Alternately or additionally,the concentration of sequencing probes in a population can be increasedto increase coverage of probes in a specific region of a target nucleicacid. The concentration of sequencing probes can be reduced to decreasecoverage of probes in a specific region of a target nucleic acid, e.g.,to above the resolution limit of the sequencing apparatus.

While the resolution limit for two detectable labels is about 600nucleotides, this does not hinder the powerful sequencing methods of thepresent disclosure. In certain aspects, a plurality of the sequencingprobes in any population will not be separated by 600 nucleotides on atarget nucleic acid. However, statistically (following a Poissondistribution), there will be target nucleic acids that only have onesequencing probe bound to it, and that sequencing probe is the oneoptically resolvable. For target nucleic acids that have multiple probesbound within 600 nucleotides (and thus are not optically resolvable),the data for these unresolvable sequencing probes may be discarded.Importantly, the methods of the present disclosure provide multiplerounds of binding and detecting pluralities of sequencing probes. Thus,it is possible in some rounds the signal from all the sequencing probesare detected, in some rounds the signal from only a portion of thesequencing probes are detected and in some rounds the signal from noneof the sequencing probes is detected. In some aspects, the distributionof the sequencing probes bound to the target nucleic acid can bemanipulated (e.g., by controlling concentration or dilution) such thatonly one sequencing probe binds per target nucleic acid.

Randomly, but in part depending on the length of the target bindingdomain, the Tm of the probes, and concentration of probes applied, it ispossible for two distinct sequencing probes in a population to bindwithin 600 nucleotides of each other.

Alternately or additionally, the concentration of sequencing probes in apopulation can be reduced to decrease coverage of probes in a specificregion of a target nucleic acid, e.g., to above the resolution limit ofthe sequencing apparatus, thereby producing a single read from aresolution-limited spot.

If the sequence, or part of the sequence, of a target nucleic acid isknown prior to sequencing the target nucleic acid using the methods ofthe present disclosure, the sequencing probes can be designed and chosensuch that no two sequencing probes will bind to the target nucleic acidwithin 600 nucleotides of each other.

Prior to hybridizing sequencing probes to a target nucleic acid, one ormore complementary nucleic acid molecules can be bound by a firstdetectable label and an at least second detectable label can behybridized to one or more of the attachment positions within the barcodedomain of the sequencing probes. For example, prior to hybridization toa target nucleic acid, one or more complementary nucleic acid moleculesbound by a first detectable label and an at least second detectablelabel can be hybridized to the first attachment position of eachsequencing probe. Thus, when contacted with its target nucleic acid, thesequencing probes are capable of emitting a detectable signal from thefirst attachment position and it is unnecessary to provide a first poolof complementary nucleic acids or reporter probes that are directed tothe first position on the barcode domain. In another example, one ormore complementary nucleic acid molecules bound by a first detectablelabel and an at least second detectable label can be hybridized to allof the attachment positions within the barcode domain of the sequencingprobes. Thus, in this example, a six nucleotide sequence can be readwithout needing to sequentially replace complementary nucleic acids. Useof this pre-hybridized sequencing probe-reporter probe complex wouldreduce the time to obtain sequence information since many steps of thedescribed method are omitted. However, this probe would benefit fromdetectable labels that are non-overlapping, e.g., fluorophores areexcited by non-overlapping wavelengths of light or the fluorophores emitnon-overlapping wavelengths of light

During sequencing, the signal intensity from a recorded color dot can beused to more accurately sequence a target nucleic acid. FIG. 21 showsexemplary imaging data recorded during a sequencing cycle of the presentdisclosure. The right panel of FIG. 21 shows the fluorescence microscopyimage recorded after a reporter probe is hybridized to the firstattachment position of a sequencing probe. Particular color dots arehighlighted and the specific color combinations recorded are noted,demonstrating that dual-fluorescence signals are clearly detectable anddistinguishable. Bright signals from fiducial makers are denoted byarrows. The left panel of FIG. 21 shows that that the spot intensity ofa particular color within a color dot can be used to determine theprobability that a specific color dot corresponds to color combinationsthat are the duplicity of one color (i.e. BB, GG, YY, or RR).

The darkening of a position within a barcode domain can be accomplishedby strand cleavage at a cleavable linker modification present within thereporter probes that are hybridized to that position. FIG. 22 depictsthe use of a cleavable linker modification to darken a barcode positionduring a sequencing cycle. The first step, depicted on the furthest leftpanel of FIG. 22, comprises hybridizing a primary nucleic acid of areporter probe to the first attachment position of a sequencing probe.The primary nucleic acid hybridizes to a specific, complementarysequence within an attachment region of the first position of thebarcode domain. The first and second domains of the primary nucleic acidare covalently linked by a cleavable linker modification. In the secondstep, the detectable labels are then recorded to determine the identityand position of a specific dinucleotide in the target binding domain ofthe sequencing probe. In the third step, the first position of thebarcode domain is darkened by cleaving the reporter probe at thecleavable linker modification. This releases the second domain of theprimary nucleic acid, thereby releasing the detectable labels. The firstdomain of the primary nucleic acid molecule, now lacking any detectablelabel, is left hybridized to the first attachment position of thebarcode domain, thereby the first position of the barcode domain nolonger emits a detectable signal and will not be able to hybridize toany other reporter probe in subsequent sequencing steps. In the finalstep, depicted in the furthest right panel of FIG. 22, a reporter probeis hybridized to the second position of the barcode domain to continuesequencing.

An attachment position of a barcode domain can be darkened by displacingany secondary or tertiary nucleic acid in the reporter probe that isbound by a detectable label while still allowing the primary nucleicacid molecule of the reporter probe to remain hybridized to thesequencing probe. This displacement can be accomplished by hybridizingto the primary nucleic acid secondary or tertiary nucleic acids that arenot bound by a detectable label. FIG. 23 is an illustrative example ofan exemplary sequencing cycle of the present disclosure in which aposition within a barcode domain is darkened by displacement of labeledsecondary nucleic acids. The far left panel of FIG. 23 depicts the startof a sequencing cycle in which a primary nucleic acid molecule of areporter probe is hybridized to the first attachment position of abarcode domain of a sequencing probe. Secondary nucleic acid moleculesbound to a detectable label are then hybridized to the primary nucleicacid molecule and the detectable label is recorded. To darken the firstposition of the barcode domain, the secondary nucleic acid moleculesbound to a detectable label are displaced by secondary nucleic acidmolecules that lack a detectable label. In the next step of thesequencing cycle, a reporter probe comprising detectable labels ishybridized to the second position of the barcode domain.

An attachment position of a barcode domain can be darkened by displacingany primary nucleic acid molecule of the reporter probe by hybridizingto the sequencing probe at the corresponding barcode domain attachmentposition nucleic acids that are not bound by a detectable label. Inthose instances where a barcode domain comprises at least onesingle-stranded nucleic acid sequence adjacent or flanking at least oneattachment position, the nucleic acid not bound by a detectable labelcan displace a primary nucleic acid molecule by hybridizing to theflanking sequence and a portion of the barcode domain occupied by theprimary nucleic acid molecule. If needed, the rate of detectable labelexchange can be accelerated by incorporating small single-strandedoligonucleotides that accelerate the rate of exchange of detectablelabels (e.g., “Toe-Hold” Probes; see, e.g., Seeling et al., “CatalyzedRelaxation of a Metastable DNA Fuel”; J. Am. Chem. Soc. 2006, 128(37),pp 12211-12220).

The complementary nucleic acids comprising a detectable label orreporter probes can be removed from the attachment region but notreplaced with a hybridizing nucleic acid lacking a detectable label.This can occur, for example, by adding a chaotropic agent, increasingthe temperature, changing salt concentration, adjusting pH, and/orapplying a hydrodynamic force. In these examples, fewer reagents (i.e.,hybridizing nucleic acids lacking detectable labels) are needed.

The methods of the present disclosure can be used to concurrentlycapture and sequence RNA and DNA molecules, including mRNA and gDNA,from the same sample. The capture and sequencing of both RNA and DNAmolecules from the same sample can be performed in the same flow cell.The left panel of FIG. 24 is an illustrative schematic of how themethods of the present disclosure can be used to concurrently capture,detect, and sequence both gDNA and mRNA from a FFPE sample.

The sequencing method of the present disclosure further comprise stepsof assembling each identified linear order of nucleotides for eachregion of an immobilized target nucleic acid, thereby identifying asequence for the immobilized target nucleic acid. The steps ofassembling uses a non-transitory computer-readable storage medium withan executable program stored thereon. The program instructs amicroprocessor to arrange each identified linear order of nucleotidesfor each region of the target nucleic acid, thereby obtaining thesequence of the nucleic acid. Assembling can occur in “real time”, i.e.,while data is being collected from sequencing probes rather than afterall data has been collected or post complete data acquisition.

The raw specificity of the sequencing method of the present disclosureis approximately 94%. The accuracy of the sequencing method of thepresent disclosure can be increased to approximately 99% by sequencingthe same base in a target nucleic acid with more than one sequencingprobe. FIG. 25 depicts how the sequencing method of the presentdisclosure allows for the sequencing of the same base of a targetnucleic acid with different sequencing probes. The target nucleic acidin this example is a fragment of NRAS exon2 (SEQ ID NO: 1). Theparticular base of interest is a cytosine (C) that is highlighted in thetarget nucleic acid. The base of interest will be hybridized to twodifferent sequencing probes, each with a distinct footprint ofhybridization to the target nucleic acid. In this example, sequencingprobes 1 to 4 (barcode 1 to 4) bind three nucleotides to the left of thebase of interest, while sequencing probes 5 to 8 (barcodes 5 to 8) bind5 nucleotides to the left of the base of interest. Thereby, the base ofinterest will be sequenced by two different probes, thereby increasingthe amount of base calls for that specific position, and therebyincreasing overall accuracy at that specific position. FIG. 26 shows howmultiple different base calls for a specific nucleotide position on thetarget nucleotide, recorded from one or more sequencing probes, can becombined to create a consensus sequence (SEQ ID NO: 2), therebyincreasing the accuracy of the final base call.

The terms “Hyb & Seq chemistry,” “Hyb & Seq sequencing,” and “Hyb & Seq”refer to the methods of the present disclosure described above.

Any of the above aspects can be combined with any other aspect asdisclosed herein.

Definitions

The terms “annealing” and “hybridization,” as used herein, are usedinterchangeably to mean the formation of a stable duplex. In one aspect,stable duplex means that a duplex structure is not destroyed by astringent wash under conditions such as a temperature of either about 5°C. below or about 5° C. above the Tm of a strand of the duplex and lowmonovalent salt concentration, e.g., less than 0.2 M, or less than 0.1 Mor salt concentrations known to those of skill in the art. The term“perfectly matched,” when used in reference to a duplex means that thepolynucleotide and/or oligonucleotide strands making up the duplex forma double stranded structure with one another such that every nucleotidein each strand undergoes Watson-Crick base pairing with a nucleotide inthe other strand. The term “duplex” comprises, but is not limited to,the pairing of nucleoside analogs, such as deoxyinosine, nucleosideswith 2-aminopurine bases, PNAs, and the like, that can be employed. A“mismatch” in a duplex between two oligonucleotides means that a pair ofnucleotides in the duplex fails to undergo Watson-Crick bonding.

As used herein, the term “hybridization conditions,” will typicallyinclude salt concentrations of less than about 1 M, more usually lessthan about 500 mM and even more usually less than about 200 mM.Hybridization temperatures can be as low as 5° C., but are typicallygreater than 22° C., more typically greater than about 30° C., and oftenin excess of about 37° C. Hybridizations are usually performed understringent conditions, e.g., conditions under which a probe willspecifically hybridize to its target subsequence. Stringent conditionsare sequence-dependent and are different in different circumstances.Longer fragments can require higher hybridization temperatures forspecific hybridization. As other factors can affect the stringency ofhybridization, including base composition and length of thecomplementary strands, presence of organic solvents and extent of basemismatching, the combination of parameters is more important than theabsolute measure of any one alone.

Generally, stringent conditions are selected to be about 5° C. lowerthan the Tm for the specific sequence at a defined ionic strength andpH. Exemplary stringent conditions include salt concentration of atleast 0.01 M to no more than 1 M Na ion concentration (or other salts)at a pH 7.0 to 8.3 and a temperature of at least 25° C. For example,conditions of 5×SSPE (750 mM NaCl, 50 mM Na phosphate, 5 mM EDTA, pH7.4) and a temperature of 25-30° C. are suitable for allele-specificprobe hybridizations. For stringent conditions, see for example,Sambrook, Fritsche and Maniatis, “Molecular Cloning A Laboratory Manual,2nd Ed.” Cold Spring Harbor Press (1989) and Anderson Nucleic AcidHybridization, 1st Ed., BIOS Scientific Publishers Limited (1999). Asused herein, the terms “hybridizing specifically to” or “specificallyhybridizing to” or similar terms refer to the binding, duplexing, orhybridizing of a molecule substantially to a particular nucleotidesequence or sequences under stringent conditions.

Detectable labels associated with a particular position of a probe canbe “readout” (e.g., its fluorescence detected) once or multiple times; a“readout” can be synonymous with the term “basecall”. Multiple readsimprove accuracy. A target nucleic acid sequence is “read” when acontiguous stretch of sequence information derived from a singleoriginal target molecule is detected; typically, this is generated viamulti-pass consensus (as defined below). As used herein, the term“coverage” or “depth of coverage” refers to the number of times a regionof target has been sequenced (via discrete reads) and aligned to areference sequence. Read coverage is the total number of reads that mapto a specific reference target sequence; base coverage is the totalnumber of basecalls made at a specific genomic position.

A “read” is a unit of sequencer output. A contiguous stretch of sequenceinformation derived from a single original target molecule. Each readhas a quality metric that associates the confidence level of the basecalls within the read. A unit of sequencer output. A contiguous stretchof sequence information derived from a single original target molecule.In Hyb & Seq, all reads are generated via multi-pass consensus.

The “readlength” is a metric describing length of sequence (in bp) fromeach read. This metric is determined by the sequencing technology.

As used in herein, a “Hyb & Seq cycle” refers to all steps required todetect each attachment region on a particular probe or population ofprobes. For example, for a probe capable of detecting six positions on atarget nucleic acid, one “Hyb & Seq cycle” will include, at least,hybridizing the probe to the target nucleic acid, hybridizingcomplementary nucleic acids/reporter probes to attachment region at eachof the six positions on the probe's barcode domain, and detecting thedetectable labels associated with each of the six positions.

The term “k-mer probe” is synonymous with a sequencing probe of thepresent disclosure. The k-mer readout is the fundamental unit of Hyb &Seq's data. A single k-mer readout is obtained from a single targetmolecule per single Hyb & Seq cycle. Multiple Hyb & Seq cycles areperformed to generate enough discrete k-mer readouts from a singletarget molecule to enable an unambiguous alignment of discrete k-mersinto a contiguous stretch of sequence

When two or more sequences from discrete reads are aligned, theoverlapping portions can be combined to create a single consensussequence. In positions where overlapping portions have the same base (asingle column of the alignment), those bases become the consensus.Various rules can be used to generate the consensus for positions wherethere are disagreements among overlapping sequences. A simple majorityrule uses the most common base in the column as the consensus. A“multi-pass consensus” is an alignment of all discrete probe readoutsfrom a single target molecule. Depending on the total number of cyclesof probe populations/polls applied, each base position within a singletarget molecules can be queried with different levels of redundancy oroverlap; generally, redundancy increases the confidence level of abasecall.

A “consensus” is when two or more DNA sequences from discrete reads arealigned, the overlapping portions can be combined to create a singleconsensus sequence. In positions where overlapping portions have thesame base (a single column of the alignment), those bases become theconsensus. Various rules can be used to generate the consensus forpositions where there are disagreements among overlapping sequences. Asimple majority rule uses the most common base in the column as theconsensus.

The “Raw Accuracy” is a measure of system's inherent ability tocorrectly identify a base. Raw accuracy is dependent on sequencingtechnology. “Consensus Accuracy” is a measure of system's ability tocorrectly identify a base with the use of additional reads andstatistical power. “Specificity” refers to the percentage of reads thatmap to the intended targets out of total reads per run. “Uniformity”refers to the variability in sequence coverage across target regions;high uniformity correlates with low variability. This feature iscommonly reported as the fraction of targeted regions covered by >20% ofthe average coverage depth across all targeted regions. Stochasticerrors (i.e., intrinsic sequencing chemistry errors) can be readilycorrected with ‘multi-pass’ sequencing of same target nucleic acid;given a sufficient number of passes, substantially ‘perfect consensus’or ‘error-free’ sequencing can be achieved.

The methods described herein can be implemented and/or the resultsrecorded using any device capable of implementing the methods and/orrecording the results. Examples of devices that can be used include butare not limited to electronic computational devices, including computersof all types. When the methods described herein are implemented and/orrecorded in a computer, the computer program that can be used toconfigure the computer to carry out the steps of the methods can becontained in any computer readable medium capable of containing thecomputer program. Examples of computer readable medium that can be usedinclude but are not limited to diskettes, CD-ROMs, DVDs, ROM, RAM,non-transitory computer-readable media, and other memory and computerstorage devices. The computer program that can be used to configure thecomputer to carry out the steps of the methods, assemble sequenceinformation, and/or record the results can also be provided over anelectronic network, for example, over the internet, an intranet, orother network.

A “Consumable Sequencing Card” can be incorporated into a fluorescenceimaging device known in the art. Any fluorescence microscope with anumber of varying features is capable of performing this sequencingreadout. For instance: wide-field lamp, laser, LED, multi-photon,confocal or total-internal reflection illumination can be used forexcitation and/or detection. Camera (single or multiple) and/orPhotomultiplier tube (single or multiple) with either filter-based orgrating-based spectral resolution (one or more spectrally resolvedemission wavelengths) are possible on the emission-detection channel ofthe fluorescence microscope. Standard computers can control both theConsumable Sequencing Card, the reagents flowing through the Card, anddetection by the fluorescence microscope.

The sequencing data can be analyzed by any number of standardnext-generation-sequencing assemblers (see, e.g., Wajid and Serpedin,“Review of general algorithmic features for genome assemblers for nextgeneration sequencers” Genomics, proteomics & bioinformatics, 10 (2),58-73, 2012). The sequencing data obtained within a single diffractionlimited region of the microscope is “locally-assembled” to generate aconsensus sequence from the multiple reads within a diffraction spot.The multiple diffraction spot assembled reads are then mapped togetherto generate contiguous sequences representing the entire targeted geneset, or a de-novo assembly of entire genome(s).

Additional teachings relevant to the present disclosure are described inone or more of the following: U.S. Pat. Nos. 8,148,512, 7,473,767,7,919,237, 7,941,279, 8,415,102, 8,492,094, 8,519,115, U.S.2009/0220978, U.S. 2009/0299640, U.S. 2010/0015607, U.S. 2010/0261026,U.S. 2011/0086774, U.S. 2011/0145176, U.S. 2011/0201515, U.S.2011/0229888, U.S. 2013/0004482, U.S. 2013/0017971, U.S. 2013/0178372,U.S. 2013/0230851, U.S. 2013/0337444, U.S. 2013/0345161, U.S.2014/0005067, U.S. 2014/0017688, U.S. 2014/0037620, U.S. 2014/0087959,U.S. 2014/0154681, U.S. 2014/0162251, and U.S. 2016/0194701 each ofwhich is incorporated herein by reference in their entireties.

EXAMPLES Example 1—Single-Molecule Long Reads Using Hyb & Seq Chemistry

The presently disclosed sequencing probes and methods of utilizing thesequencing probes is conveniently termed, Hyb & Seq. This term isutilized throughout the specification to describe the disclosedsequencing probes and methods. Hyb & Seq is a library-free,amplification-free, single-molecule sequencing technique that usesnucleic acid hybridization cycles of fluorescent molecular barcodes ontonative targets.

Long reads using Hyb & Seq are demonstrated on a single molecule DNAtarget 33 kilobases (kb) long with the following key steps: (1) long DNAmolecules are captured and hydro-dynamically stretched onto thesequencing flow-cell; (2) multiple perfectly matched sequencing probeshybridize across the long single molecule target; (3) fluorescentreporters hybridize to the barcode region in the sequencing probes toidentify all the bound sequences; and/or (4) relative positions ofsequences within a single molecule target are determined usingspatially-resolved fluorescence data.

Key advantages of long reads using Hyb & Seq, include but are notlimited to: read lengths determined by molecule length, not limited bychemistry; simple, limited sample preparation results in lessfragmentation; positional information associated with sequencing probesaids assembly; and/or capability to phase variants into long-rangehaplotypes.

Hyb & Seq Chemistry Design—Sequencing Probes comprise a target bindingdomain that base-pairs with a single molecule target and a barcodedomain having at least three positions (R₁, R₂, and R₃) that correspondto the hexamer sequence present in the target binding domain. A set of4096 sequencing probes enables sequencing of any target sequence.Reporter Probes: Three reporter probes bind sequentially to thepositions of the barcode domain. Each reporter complex corresponds to aspecific dinucleotide. Hybridization drives the functionality.

Long read and short read sequencing methods of the present disclosureuse the same simple probe hybridization workflow for targeted capture ofnucleic acids as shown in FIGS. 18 and 19. A plurality of sequencingprobes can hybridize to a target nucleic acid concurrently, as shown inthe right panel of FIG. 18, and optical resolution allows several spotsper long target to be individually distinguished, as shown in the rightpanel of FIG. 19. By hybridizing and recording a plurality of sequencingprobes concurrently, the information content of a single read isincreased. Long-range haplotypes are inherent in single-moleculeanalysis and can be assembled by actual physical location rather thancomputational reconstruction. Long sequencing reads up to hundreds ofkilobases are feasible using the sequencing methods of the presentdisclosure.

Pools of specific sequencing probes hybridize to 15 kilobase targets inexpected patterns as shown in FIG. 20. Sequencing probes hybridize tostretched targets (preferably hydro-dynamically stretched targets) atexpected sequence-specific positions and relative physical distances.The sequencing methods of the present disclosure have increasedinformation content compared to short-read technologies, allowing morebases to be read out each cycle. The sequencing methods of the presentdisclosure also record the relative position of sequencing readouts,which aids in assembly of long reads. Using the sequencing methods ofthe present disclosure, read length=consensus sequence length=length ofcaptured target molecule.

FIG. 27 shows the results of an experiment in which 33 kilobase DNAfragments were captured, stretched, hybridized to sequencing probes andreporter probes, and detected. The sequencing methods of the presentdisclosure are compatible with DNA fragments up to 33 kilobases andbeyond. Read length is limited only by initial length of the targetnucleic acid fragment, not enzymes or sequencing chemistry.

FIG. 13 shows additional capabilities of the sequencing method of thepresent disclosure with respect to targeted phased long reads.Long-range phased haplotypes are inherent in data and easily identifiedfor phasing of variants. Sequencing of the entire long target moleculeis not necessary as “blocker oligos” can be used to limit sequencecycling to sequencing windows of interest.

The results of Example 1 show that the sequencing method of the presentdisclosure is capable of single molecule sequencing with long readlengths. In particular, the results show: successful capture andhydro-dynamic stretching of a 15 kilobase and 33 kilobasesingle-stranded DNA molecule; spatially-resolved fluorescence dataaccurately corresponds to the actual relative positions across the longsingle molecule; and simultaneous readout of 10+ base sequences persequencing cycle.

Example 2—ShortStack™ Technology: Accurate, Reference-Guided Assembly ofHyb & Seq Reads for Targeted Sequencing to Resolve Short NucleotideVariants and InDels

ShortStack is an open source algorithm designed to perform assembly ofHyb & Seq's unique hexamer readouts (hexamer spectra). The algorithm isa statistical approach to target identification utilizing hexamer readsfrom each imaged feature and to perform assembly of hexamer readoutsinto a consensus sequence on a single molecule basis witherror-correction.

Single molecule sequencing using Hyb & Seq chemistry and ShortStack wasperformed as follows: hexamer readout of the single molecule target wasgenerated after each cycle of hybridization using Hyb & Seq chemistry;after many cycles of hybridization, hexamer spectra that cover eachsingle molecule target regions were produced; and hexamer spectra areused with a reference sequence of each of the target nucleic acidmolecules to derive the consensus sequence of each single-moleculetarget.

The results of target sequencing using Hyb & Seq technology withShortStack show: single molecule target identification algorithm usingthe hexamer spectra had 100% success rate; reference guided assemblyalgorithm produced single molecule consensus accuracy of >99% (^(˜)QV32) at 5× coverage; concordant somatic variant detection (R^(2˜)90%) wasdemonstrated using a pre-characterized reference gDNA sample; and/or insilico experiments using all hexamers and ShortStack confirmed averageQV>90 across larger target panels The ShortStack algorithm canaccurately assemble Hyb & Seq data. FIG. 28 shows the results from asequencing experiment obtained using the sequencing method of thepresent disclosure and analyzed using the ShortStack algorithm. In thisexperiment, the target nucleic acids that were sequenced includedfragments of the genes BRAF (SEQ ID NO: 3), EGFRex18 (SEQ ID NO: 4),KRAS (SEQ ID NO: 5), PIK3CA (SEQ ID NO: 6), EGFRex20 (SEQ ID NO: 7) andNRAS (SEQ ID NO: 8). FIG. 28 shows both the base coverage and variantcalling. The coverage plots show coverage of bases in FFPE(formalin-fixed paraffin-embedded) gDNA. The results show that mostbases across a variety of targets are covered by available sequencingprobes. The error plots show error rate vs coverage at queried positionin FFPE gDNA samples across a variety of targets. The results show thatat 8× coverage, error rates are <1%. The frequency plot shows thecorrelation between expected and known frequency of variants insequenced Horizon gDNA samples. The table provides sequenced HorizonGenomic Reference gDNA and shows that the fraction of variant moleculesis consistent with known frequency of reference sample.

The results in Example 2 show that ShortStack is an accurate algorithmfor sub-assembly of hexamer spectra obtained using the sequencing methodof the present disclosure. In particular the results show: 100% accuracyin target identification and average per-base quality values >30 usingsimulated data; at 5× coverage, >99% accuracy in base calling inexperimental Hyb & Seq data; detection of variants from genomic DNA atfrequencies consistent with known values (R^(2˜)90%), and computationalperformance is efficient and scales linearly with the number of hexamersassembled, capable of assembling 69k molecules in ˜15 min on a personalcomputer.

Example 3—Library-Free, Targeted Sequencing of Native gDNA from FFPESamples Using Hyb & Seq™ Technology—the Hybridization Based SingleMolecule Sequencing System

A targeted cancer panel sequencing of native gDNA from FFPE samplesusing the sequencing method of the present disclosure (Hyb & Seq) wasperformed to demonstrate: targeted single-molecule sequencing ofoncogene targets with accurate base-calling; accurate detection of knownoncogenic Single Nucleotide Variants (SNVs) and Insertions/Deletions(InDels); multiplexed capture of oncogene targets from FFPE-extractedgDNA (median DNA fragment size 200 bases); and/or end-to-end automatedsequencing performed on an advanced prototype instrument.

Hyb & Seq chemistry and workflow were demonstrated as follows: genomictargets of interest are directly captured onto the sequencing flow cell;a pool containing hundreds of hexamer sequencing probes is flowed intothe sequencing chamber; fluorescent reporter probes sequentiallyhybridize to the barcode region of the sequencing probe to identify thehexamer bases over 3 reporter exchange cycles; once the bases areidentified, the sequencing probe is washed away; and the cycle isrepeated with a new pool of sequencing probes until the target regionshave been read to sufficient depth

Key Advantages of Hyb & Seq: simple and rapid FFPE workflow—Clinicalspecimen to start of sequencing within 60 minutes; no enzymes oramplification/No library construction; 15 minutes of totalhands-on-time; high accuracy—Low chemistry error rate+intrinsic errorcorrection; and/or both long & short reads—Read length defined by inputsample, not limited by chemistry.

Hyb & Seq Chemistry Design is as described in Example 1. Hyb & Seqsample preparation for processing FFPE tissues consists of three simplesteps: (1) Single-tube deparaffinization and lysis; (2) Removal ofparticulates using a syringe filter; and (3) Optional DNA fragmentationand target capture. The process requires one to three 10 micron FFPEcurls used per sample. The entire process is completed within 60 minutesand it needs only common lab equipment: heat block, pipette, filter andreagents.

FIG. 29 shows a schematic illustration of the experimental design forthe multiplexed capture and sequencing of oncogene targets from a FFPEsample. A total of 425 sequencing probes were designed and constructedto sequence portions of 11 oncogenic gene targets (SEQ ID NOs: 3-13).The loci of known variant for each gene target was covered with manysequencing probes (perfect match+single mismatch). Base coverage andbase by base accuracy was measured across these regions. Using apre-characterized reference sample, accuracy of variant detection wasobtained. The top panel of FIG. 29 shows that sequencing Probes (blue)align to a target sequence (grey) surrounding a known variant location(red). For each variant location (red), 4 probe sequences were includedwith each (A, G, C, T) base variant. During Sequencing, single targetDNA molecules were tracked for 800 barcode exchange cycles, providingmultiple hexamer reads which are reassembled by the ShortStack™algorithm, as described in Example 2.

FIG. 28 shows the sequencing results including the average coverage ofeach target, the single base error rate, and the observed vs. expectedvariant frequencies. The results in Example 3 show that Hyb & Seqsequencing can be used to perform multiplexed sequencing of 11 targetregions in FFPE and reference gDNA samples with Single nucleotidevariations detected with low error.

Example 4—Direct Single-Molecule RNA Sequencing without cDNA ConversionUsing Hyb & Seq Chemistry

Direct single-molecule RNA sequencing using Hyb & Seq chemistry wasdemonstrated as follows: native RNA molecules were captured directlywithout cDNA conversion and immobilized onto sequencing flow cell; apool containing hundreds of hexamer sequencing probes was flowed intothe sequencing flow cell; a perfectly matched sequencing probe washybridized randomly on a single molecule RNA target; fluorescentreporter probes were sequentially hybridized to barcode region ofsequencing probe to identify hexamer bases; and bases were identifiedand then sequencing probes washed away; cycle was repeated until targethad been read to sufficient depth.

Key results: targeted single-molecule RNA was sequenced showing similarcoverage profiles to DNA; RNA molecules were stably maintained on theflow cell throughout more than 200 Hyb & Seq cycles; mRNA and genomicDNA were simultaneously captured and quantitated from a single FFPEslice; and/or eight transcripts were multiplex captured and quantitatedusing as little as 10 ng of total RNA.

Hyb & Seq Chemistry Design is as described in Example 1. The left panelof FIG. 30 shows an illustrative schematic of the experimental stepsassociated with direct RNA sequencing compared to the steps associatedwith conventional RNA sequencing performed using cDNA conversion. Themiddle and left panels of FIG. 30 show results from experiments to testthe compatibility of RNA molecules with the sequencing method of thepresent disclosure. In the experiment, 4 target RNA molecules weresequenced (SEQ ID NOs: 14-17). The results show that RNA molecules canbe captured and detected for at least 200 sequencing cycles,demonstrating the compatibility of the sequencing methods of the presentdisclosure and RNA molecules.

FIG. 31 shows the results from an experiment to validate directsingle-molecule RNA sequencing using the sequencing method of thepresent disclosure. Native RNA molecules encoding a fragment of NRASex2(SEQ ID NO: 18) were captured directly without cDNA conversion andimmobilized onto a sequencing flow cell and sequenced using the presentmethods. The experiment was also repeated using captured DNA moleculesinstead of RNA. FIG. 31 shows that sequencing coverage for DNA and RNAwas comparable, demonstrating that RNA can be directly sequenced withoutconversion to cDNA using the sequencing method of the presentdisclosure.

FIG. 24 shows an example of integrated capture of RNA and DNA from aFFPE sample. The left panel of FIG. 24 is an illustrative schematic ofhow the methods of the present disclosure can be used to concurrentlycapture, detect, and sequence both gDNA and mRNA from a FFPE sample.Sample are prepared using the same FFPE workflow described in Example 3.The same capture protocol is used, but with RNA- and DNA-specificcapture probes. The DNA and RNA molecules are concurrently sequenced inthe same flow cell with the same sequencing probes. The right panel ofFIG. 24 shows the results from an experiment to concurrently capture anddetect NRAS RNA and DNA from a tonsil FPPE sample. The fluorescenceimage shows that both RNA and DNA can be captured and detected. The bargraph demonstrates that specific RNA and DNA capture probes are requiredto concurrently capture RNA and DNA.

FIG. 32 shows the results of a multiplex target capture of an RNA panel.Multiplex capture of 8 mid-to-high expressing transcripts on HumanUniversal Reference RNA with various input amounts of total RNA (0 ng, 1ng, 10 ng, 100 ng, 1000 ng) was performed. Multiplexed captured RNAmolecules were immobilized onto a flow cell and specific sequencingprobes and reporter probes were hybridized to the immobilized RNAmolecules for quantitation. The bottom panel of FIG. 32 shows afluorescence image from 100 ng input capture. One example of each RNA iscircled with transcript name and corresponding reporter complex colorcombination used for identification. The top panel of FIG. 32 shows thequantitation of counts for each specific RNA target.

The results in Example 4 show that single-molecule RNA sequencing isachieved with Hyb & Seq chemistry. In particular, the resultsdemonstrate: (1) direct RNA sequencing without cDNA conversion; (2) RNAmolecules are stable throughout the Hyb & Seq cycling process; (3) bothRNA and DNA molecules can be captured and sequenced in one Hyb & Seqworkflow; and (4) target capture of mRNA panel can be performed with aslittle as 10 ng of total RNA input.

Example 5—Integrative Bioinformatics Algorithm for High ThroughputMolecule-Level Short-Reads Generated from Hyb & Seq Sequencing Platform

The ShortStack™ software is designed to perform standardsequencing-based bioinformatics analysis tasks such as alignment,error-correction, mutation-calling, and read assembly. FIG. 33 shows aschematic overview of the steps of the ShortStack™ software pipeline,including: alignment of hexamers and coverage estimation; mutatedsequence identification; graph data structure construction; and/ormolecule level sequence reconstruction and error correction.

All algorithms were performed strictly within the information obtainedfrom a single molecule, ensuring that the final mutation call resultswere not biased by the mutation frequency of the sample. Hexamers weregrouped into different molecules according to the panel bindingposition. To assign molecules to targets, the hexamers were aligned permolecule to all different target regions and the top matched gene targetwas selected.

A statistical metric was measured to assess the quality of the moleculeidentification. The alignment against N different target regionsproduces a distribution of N summed coverage values for each target. Thetop summed coverage score match was selected as the correct match.Z-score statistics of the selected top match score against the scoredistribution of all N different targets were measured. Low confidencemolecule identifications (under z-score of 2.5 sigma) were filtered out.

Key Advantages of ShortStack™ algorithm include: accurately handlespossible sequence ambiguities by implementing a hierarchical hash indexdesign; and/or advanced algorithm design structure assures the mappingquality by prioritization and prevents the overestimation of mutations.

In addition, the mutation graph data structure enables computationalmodeling of various types of mutations (substitution, insertion, anddeletion) and produces output for sequence reconstruction and variantcalling: substitution variants are represented as additional nodes inthe graph of same length with the original sequence; insertions can bemodeled by adding any length of connected nodes; deletions are modeledas adding an artificial node in the graph with empty base pair string;in a blind mutation search (i.e. a search for mutation tolerant sequencealignments), hamming distances are measured from every referencesequence position and new nodes are added to the graph representingsearched mutations; and/or coverage estimation for mutated hexamers isperformed using the hierarchical hash table.

The constructed graph data structure enables molecule level sequencereconstruction and instrument error correction. In the constructedgraph, the dynamic programming algorithm was applied to find the bestscoring path where the score was defined as the normalized basecoverages. The best scoring path of the graph represented the moleculelevel sequence reconstruction. The correct mutated sequences wereincluded, while the instrument errors in hexamers were discarded.

Simulated data sets confirmed that the software was able deliver highlyaccurate molecule level sequence assembly and mutation calling results.FIG. 34 shows the results of mutational analysis of simulated data setsusing the ShortStack™ software pipeline. These results show the mutationcalling accuracy for 10 random mutations. In medium instrument errordatasets, the accuracy showed 99.39% (targeted search) and 98.02% (blindsearch) on average. Under the elevated instrument error simulations, theperformance showed 97.19% (targeted search) and 93.53% (blind search) onaverage. When the molecule level base coverage threshold was increasedto 2×, results improved to 99.5% (2× coverage) and 99.9% (3× coverage).

The ShortStack™ software can process a broad scope of various mutations.FIG. 35 shows the overall mutation calling accuracy, where each barrepresents averaged value from 10 different mutated targets acrossvarious types of mutations. The length of insertions and deletions isselected between 1 bp and 15 bp. These results show that the mutationcalling accuracy was 94.4% (1× coverage), 97.7% (2× coverage), to 98.5%(3× coverage).

Example 6—Sample Preparation for Processing FFPE Tissue for Hyb & Seq

Formalin-fixed paraffin embedded (FFPE) tissue is a challenging sampleinput type for traditional sequencing platforms. Hyb & Seq's samplepreparation methods successfully process FFPE tissue inputs fordownstream sequencing. First, the nucleic acid(s) to be sequenced isextracted from formalin-fixed, paraffin embedded (FFPE) tissue in asingle-step process. One or more 10 μm thick FFPE curl is heated in anaqueous-based nucleic acid extraction buffer to simultaneously melt theparaffin wax, decompose the tissue, and release nucleic acid from thecells. Suitable extraction buffers are known in the art and typicallyinclude proteinases, detergents such as Triton-100, chelating agentssuch as EDTA, and ammonium ions. The FFPE curl and extraction buffer areincubated at 56° C. for 30 minutes to separate the paraffin from thetissue and allow the Proteinase K to digest the tissue structure andexpose the embedded cells to the detergent to enable cell lysis. Thesolution is inverted three times at 8 minute intervals to assist inmixing of the reagents during the tissue deparaffinization and digestionprocess. Following this step, the solution is heated to 98° C. tofacilitate the reversal of the formaldehyde cross-links to furtherassist in the extraction of nucleic acids.

Once the nucleic acids have been extracted from the FFPE tissue, thesolution is filtered using a glass fiber filter with 2.7 μm pore size(Whatman) to remove tissue debris and congealed paraffin. The resultingsolution is a homogenous, semi-opaque solution containing nucleic acidswhich are highly fragmented due to the formalin-fixation process andstorage conditions. If further fragmentation is required, the DNA can bemechanically sheered using a Covaris focused-ultrasonicator. Due tobuffer conditions, extended sonication is required to shear the nucleicacids. Sonicating using the standard settings of 50 W peak incidentpower, 20% duty factor, 200 cycles/burst were used for 600 seconds toachieve the maximal increase in targets captured. To achieve shorterfragment length, emulsified paraffin can be precipitated out of thefiltered solution by centrifuging at 21,000 g and 4° C. for 15 minutes.This allows the DNA to be sheared down to about 225 bp.

Next, target capture is performed by binding pairs of capture probes totarget nucleic acid molecules during a rapid hybridization step. The 5′capture probe contains a 3′ biotin moiety, which allows the target tobind to a streptavidin-coated flow cell surface during the targetdeposition process. The 3′ capture probe contains a 5′ tag sequence(G-sequence) that enables binding to beads during the purificationprocess. The reaction rate is driven by the capture probe concentrationwhich are added in the low nanomolar range to maximize the reactionrate. The capture probes hybridize to the target in a manner that flanksto region of interest in order to generate a sequencing window. For eachDNA target, the capture probe set also includes an oligo composed of thesame sequence as the sequencing window to hybridize to targets'antisense strand and prevent reannealing. The solution containing thecapture probes is heated to 98° C. for 3 minutes to denature the genomicDNA, followed by a 15-minute incubation at 65° C. The concentration ofNaCl in the range of 400 mM to 600 mM is used for this hybridizationreaction. A panel of over 100 targets that have been experimentallyvalidated is listed in the Table 3, detailing the gene and exon of thetargeted DNA region.

TABLE 3 Gene and Exon of targeted DNA regions Gene Target ABL1 ABL1_ex4ABL1_ex6 ABL1_ex7 AKT1 AKT1_ex6 ALK ALK_ex26 APC APC_ex5 APC_ex16APC_ex17 APC_ex17 APC_ex17 APC_ex17 APC_ex17 ATM ATM_ex8 ATM_ex9ATM_ex11 ATM_ex26 ATM ATM_ex34 ATM_ex39 ATM_ex49 ATM_ex49 ATM_ex55ATM_ex59 BRAF BRAF_ex8 BRAF_ex11 BRAF_ex13 BRAF_ex15 CDH1 CDH1_ex9 CSF1RCSF1R_ex3 CSF1R_ex22 CTNNB1 CTNNB1_ex3 CTNNB1_ex6 CTNNB1_ex16 EGFREGFR_ex3 EGFR_ex10 EGFR_ex15 EGFR_ex18 EGFR_ex20 EGFR_ex21 ERBB2ERBB2_ex7 ERBB4 ERBB4_ex4 ERBB4_ex5 ERBB4_ex7 ERBB4_ex8 ERBB4_ex23ERBB4_ex25 EZH2 EZH2_ex8 EZH2_ex11 EZH2_ex15 FBXW7 FBXW7_ex2 FBXW7_ex5FBXW7_ex7 FBXW7_ex8 FBXW7_ex9 FBXW7_ex10 FGFR1 FGFR1_ex6 FGFR2 FGFR2_ex7FLT3 FLT3_ex11 FLT3_ex12 FLT3_ex21 GNAQ GNAQ_ex5 IDH1 IDH1_ex4 IDH1_ex10IDH2 IDH2_ex4 JAK2 JAK2_ex3 JAK2_ex7 JAK2_ex14 JAK2_ex20 KDR KDR_ex7KDR_ex7 KDR_ex9 KDR_ex11 KDR_ex27 KDR_ex30 KIT KIT_ex5 KIT_ex9 KIT_ex14KIT_ex14 KIT_ex17 KIT_ex18 KRAS KRAS_ex2 KRAS_ex3 KRAS_ex4 MEK MEK_ex3MET MET_ex2 MET_ex3 MET_ex11 MET_ex14 MET_ex16 MET_ex19 MLH1 MLH1_ex12MLH1_ex16 NOTCH1 NOTCH1_ex26 NRAS NRAS_ex2 NRAS_ex3 NRAS_ex3 NRAS_ex4PDGFRA PDGFRA_ex1 PDGFRA_ex4 PDGFRA_ex7 PDGFRA_ex10 PDGFRA_ex11PDGFRA_ex14 PDGFRA_ex15 PDGFRA_ex16 PDGFRA_ex18 PDGFRA_ex23 PIK3CAPIK3CA_ex2 PIK3CA_ex3 PIK3CA_ex7 PIK3CA_ex10 PIK3CA_ex14 PIK3CA_ex21PIK3CA_ex21 PTEN PTEN_ex5 PTEN_ex7 PTEN_ex8 PTENP1 PTENP1_ex1 RB1RB1_ex10 RB1_ex17 RB1_ex17 RB1_ex20 RB1_ex22 RET RET_ex12 RET_ex15 SMAD4SMAD4_ex3 SMAD4_ex8 SMAD4_ex9 SMAD4_ex10 SMAD4_ex11 SMARCB1 SMARCB1_ex5TP53 TP53_ex4 TP53_ex6

After the targeted DNA regions are bound with capture probes, they arepurified from the rest of the genomic DNA to create an enriched solutionof the targets. Beads coated with the anti-sense oligo (anti G-sequence)to the 3′ capture probes' binding sequence are incubated with thecapture reaction mix for 15 minutes at room temperature. After thebinding step, the beads are washed three times with 0.1×SSPE to removenon-target DNA and the biotin-containing 5′ capture probes. Followingthe washes, the beads are re-suspended in 14 μL of 0.1×SSPE then heatedat 45° C. for 10 minutes to elute the purified DNA targets from thebeads. After elution, 1 μL of 5 M NaCl is added to ensure the captureprobes remain bound to the DNA targets.

The final step of the sample preparation process is the deposition ofthe DNA targets onto the flow cell surface, where they can be analyzedusing the probes of the present disclosure as disclosed herein. Asyringe pump is utilized to control the rate at which the targets areloaded into the flow cell fluidic channel, such that all targets havetime to diffuse across the height of the channel and bind to thestreptavidin surface. This method of loading generates a densitygradient of targets, where the highest number of molecules per unit areais greatest at the fluidic channel inlet and decreases along the channellength in the direction of the fluidic flow towards the outlet. A flowrate of 0.35 μL/second achieves a quantitative capture within a channellength of about 10 mm for a channel width of 1.6 mm and height of 40 μm.Once the targets are bound to the surface by the biotinylated 5′ captureprobe, a solution of biotinylated oligo (G-hooks) that are the reversecomplement of the 3′ capture probes' bind sequence are injected to pindown the free end of the targets to create a bridged structure, wherethe ssDNA region in the middle is the sequencing window of interest.Next, a solution of G-sequence oligos are added to hybridize to theexcess G-hooks on the surface to reduce the amount of ssDNA on thesurface. FIG. 11 shows the capture of a target nucleic acid using a twocapture probe system of the present disclosure.

Example 7—Multi-Color Reporter Image Processing for Hyb & Seq

The image processing pipeline includes the following steps backgroundsubtraction, registration, feature detection, and classification. Inbackground subtraction, the mean background of any given channel is afunction of shot noise and exposure. In our system, the blue channel hasthe highest background levels coupled with greater variance. A simpletophat filter with a circular structuring element of radius 7 pixels isapplied to perform localized background subtraction. For registration,it is imperative that the features of interest as perfectly aligned formulti-color and multi-cycle feature analysis. This system requires twoforms of registration. For the first form, a local affine transformationis applied to all image channels within a single acquisition stack. Thistransformation is a function of the optical system and hence isconsistent for a given instrument. This function is computed in advancefor every run and is applied to every image acquired. For the secondform, a global transformation in the form of a rigid shift is computedusing normalized cross-correlation to capture drift of the mechanicalgantry during the run. The next step is feature detection.

Once all the images are registered, features are detected using amatched filter via a LoG (Laplace of Gaussian) filter. The filter isapplied with a fixed kernel size (matched to the diffraction limit ofthe features) and a varying standard deviation (matched to thewavelength of the corresponding channel) to match to enhance spotresponse. Local maxima are used to identify potential reporterlocations. The associated intensity values for each identified featureare retrieved for classification. The final step is classification. Themulti-color reporter intensities are classified using the Gaussiannaîve-Bayes model. The model assumes that the reporter intensities areindependent and follow a normal distribution. The model then calculatesthe probability that a specific feature 9 (specified by intensities inall channels {tilde over (x)}₁) belongs to a certain class (C_(k)) usinga maximum a posteriori or MAP rule:

$\hat{y} = {{argmax}_{\; {\{{k \in {\{{1,{\ldots \; K}}\}}}\}}}{p\left( C_{k} \right)}{\prod\limits_{i = 1}^{n}{p\left( x_{i} \middle| C_{k} \right)}}}$

The intensity distributions for a reporter complex labeled with aparticular color combinations is shown in FIG. 36. FIG. 36 illustratesthe coding scheme using 2 dyes: blue and red. There are six classes(including background) possible in a 2-color coding scenario. In theimplemented system, the choice of four colors results in 14 potentialclasses. Note that there is some overlap between the single half dye vsfull dye distributions. Consequently, classification between theseclasses presents a higher error rate, with a maximum miss-classificationrate of 11.8% between ‘xG’ and ‘GG’. The miss-classification rates forthe 10 Class model is less than 0.2%. Since each reporter requires amaximum of eight classes, it is simple to choose the ones with leastclassification error. The detected color code is translated into anidentified base pair based on a look up table. Using the probes of thepresent disclosure as disclosed herein, a feature is tracked acrossmulti-cycles.

Example 8—Target Nucleic Acid Purification and Deposition Using CaptureProbes

To capture target nucleic acid molecules, a two capture probe system isused for highly specific enrichment. Capture probes are designed to bindto the target nucleic acid at positions flanking the targeted region ofinterest, creating a “sequencing window”. The 5′ capture probe, referredto as CapB, contains a 3′ biotin moiety. The 3′ capture probe, referredto as CapA, contains a 5′ affinity tag sequence, referred to as theG-sequence. On average, capture probes are approximately 40 nucleotidesin length and designed based on Tm and sequence context. Sequencingwindows are around 70 nucleotides in length and are easily adjusted.FIG. 11 shows a schematic of the two capture probe system.

The biotin moiety on CapB tethers the target nucleic acid to astreptavidin-coated flow cell surface for sequencing. The affinity tagon CapA allows for the reversible binding of target nucleic acidmolecules to magnetic beads during purification. The use of CapA andCapB allows for highly stringent target enrichment since both probesremain bound to a single target nucleic acid molecule in order for thattarget to survive both the magnetic bead purification and the surfacedeposition process. Multiplexed capture has been demonstrated with up to100 targets at once. In order to achieve an efficient capture within ashort period of time, capture probes are added at the concentrationrange of 1 nM to 10 nM.

In experimental tests, a panel of ˜10 target nucleic acid molecules werepurified using G-beads and the two probe capture system. CapA and CapBprobes were first hybridized to target nucleic acids. The G-sequenceportion of the bound CapA probes were then hybridized to the G-hooks onthe G-beads, thereby linking the target nucleic acid molecule to theG-beads. A series of stringent washes using 0.1×SSPE was performed toremove non-targeted DNA and unbound CapB. To release the target nucleicacid molecules from the G-beads, a low-salt, 45° C. elution wasperformed to denature the G-sequence while still permitting CapA andCapB to remain hybridized to the target nucleic acid.

Tests show that when purifying a panel of ˜100 target nucleic acidmolecules, the non-specific/background signal increases significantly.This increase in background could be due to several factors including:(1) increased interactions between CapA and CapB probe species, whichleads to increased amounts of free CapB probe carried throughpurification; and (2) increased interaction between CapB probes and theG-hooks or the G-beads, which leads to the purification of unwantedtarget nucleic acids. Furthermore, as the size of the panel increases,the possible interactions between CapB species, CapA species, andsequencing probes increase exponentially. These interactions caninterfere with the ability to densely deposit targets and lead to wastedsequencing reads.

To reduce non-specific and background signal due to the purification offree probe species and unwanted target nucleic acid molecules, severalmodifications to the purification procedure can be made. First, theinclusion of formamide at a concentration of 30% v/v in the buffer usedduring the binding of target nucleic acid molecules to G-beads decreasesbackground counts by two-fold (as measured by counts in controls lackingtarget molecules), likely through interfering with imperfecthybridizations of free capture probe with G-hooks, allowing excessprobes to be washed away. Secondly, the inclusion of four iso-dG basesin the G-hook on the G-beads (iso-G-hooks) and the complementary iso-dCbases in the CapA G-sequence decreases background counts by three-fold(as measured by counts in controls lacking target molecules). Iso-dC andiso-dG are isomer variants of the natural dC and dG bases. Since, isobases will base-pair with other iso bases but not natural bases,imperfect hybridization between capture probes and iso-G-hooks can onlyexist between the non-iso bases of the G-sequence and iso-G-hooks. Theseimperfect interactions are more easily disrupted during stringentwashes. Finally, subsequent purification of the iso-G-bead eluates withAmpure® XP (Agencourt Biosciences Company) beads further decreasesbackground counts by at least 20-fold (as measured by counts in controlslacking target molecules). During, Ampure® XP bead purification, a DNAsample is mixed with a suspension of carboxylated magnetic beads in asolution of polyethylene glycol (PEG) and NaCl. The concentration of PEGand NaCl can be titrated such that only molecules above a molecularweight threshold precipitate and bind to the beads. Hyb & Seq targetshybridized to capture probes are on the order of 81 kDa, while freeprobes are on the order of 17 kDa or less. By mixing the Ampure® XP beadsuspension with iso-G-bead eluate at a volume ratio of 1.8:1, hybridizedtargets are bound to the beads and a significant portion of free probescan be washed away before the final elution.

Thus, a model purification workflow consists of the following steps: (1)Hybridization of capture probe-target nucleic acid assemblies toiso-G-beads in 5×SSPE/30% formamide; (2) Washing of the iso-G-beads with0.1×SSPE; (3) Elution of capture probe-target nucleic acid assemblies at45° C. in 0.1×SSPE; (4) Binding of iso-G bead eluates to a 1.8× volumeof Ampure® XP beads; (5) Washing of Ampure® XP beads with 75% ethanol;and (6) Elution of capture probe-target nucleic acid assemblies in0.1×SSPE, such that the targets are eluted in 7.5 followed by theaddition of 0.5 μL of 5 M NaCl.

After purification, capture probe-target nucleic acid assemblies aredeposited onto the sequencing surface using an infusion syringe pump toslowly inject the purified targets through the flow cell. To determinethe deposition gradient, the flow cell is imaged at various positionsalong the channel length. A typical deposition gradient is shown in FIG.37. For a channel height of 20 μm, loading the sample at a flow rate of0.167 μL/min will concentrate the targets such that 80% of all targetsbind within 5.1 mm along the channel length, which represents ˜240 FOVsfor the Gen2 imager with a FOV of 0.0357 mm² and flow cell channel widthof 1.7 mm. The gradient can be modulated by adjusting the flow rateduring deposition.

The procedures described above were used to test the purification anddeposition of a 100plex target nucleic acid panel with genomic DNAsheared to a size of ˜300 base pairs. A series of experiments wasperformed in triplicate with a range of DNA input between 25 ng and 500ng. The total number of targets on the flow cell was extrapolated byimaging the deposition gradient to obtain the number of average counts,as shown in FIG. 38. The capture efficiency was 6.6% and was consistentover the range of DNA mass inputs.

Example 9—Design and Features of Sequencing Probes

Sequencing probes hybridize to a target nucleic acid molecule via atarget binding domain. In the present example, the target binding domainis 8 nucleotides long and contains a locked nucleic acid (LNA) hexamerthat is flanked by (N) bases that can be a universal/degenerate base ora canonical base (N₁-B₁-B₂-B₃-B₄-B₅-B₆-N₂, where B₁ to B₆ are LNAs andN₁ and N₂ are universal/degenerate bases or a canonical base that isindependent of the nucleic acid sequence of the (6-mer) sequenceB₁-B₂-B₃-B₄-B₅-B₆). A complete set of 4,096 sequencing probes encodesall possible hexamers and enables sequencing of any target nucleic acid.Each sequencing probe also includes a barcode domain that encodes forthe hexamer sequence present in the target binding domain. Each barcodedomain contains three positions (R₁, R₂, and R₃). Each position in thebarcode domain corresponds to a specific dinucleotide in the hexamer ofthe target binding domain and contains a unique sequence that can bindto a specific labeled reporter complex. A schematic overview ofsequencing probes are shown in FIG. 1. Each position in the barcodedomain encodes eight “color combinations”, created using fourfluorescent dyes: blue (B); green (G); yellow (Y); and red (R). Duringeach cycle of sequencing, a reporter complex is bound to one of thethree positions in the barcode domain, indicating the identity of thecorresponding dinucleotide in the hexamer of the target binding domain.During three sequential sequencing cycles, three “color combinations”are recorded, one for each position in the barcode domain, allowing forthe identification of the entire hexamer of the target binding domain.The 4,096 sequencing probes are split into 8 pools and each isassociated with one of 512 possible barcodes.

Example 10—Reporter Complex Design, Purification, and Binding Conditions

In this example, each reporter complex is a 37 DNA oligomer branchedstructure designed to hold a total of 30 fluorescent dyes, with 15 dyesfor each color of the color combination. The 37 DNA oligomers that makeup the reporter complex can be classified by their size. The largestoligomer, called the primary nucleic acid, is covalently attached to acomplementary nucleic acid that is either 12 or 14 nucleotides inlength. The primary nucleic acid is 96 nucleotides long. The primarycomplementary nucleic acid binds to positions R₁, R₂, or R₃ on thebarcode domain of the sequencing probe. The next largest DNA oligomersare 89 nucleotides long and are called secondary nucleic acids. Thereare six secondary nucleic acids per reporter complex, with threesecondary nucleic acids per for each color of the color combination.Each secondary nucleic acid comprises a 14 nucleotide long sequencesthat allows the secondary nucleic acids to hybridize to the primarynucleic acid. The smallest DNA oligomers are 15 nucleotides long and arecalled the tertiary nucleic acids. There are 30 tertiary nucleic acidsper two color probe, with 15 tertiary nucleic acids per color. Fivetertiary nucleic acids bind to each secondary nucleic acid. A schematicof the 37 DNA oligomer branched structure is shown in FIG. 4.

The tertiary nucleic acids include a detectable label in the form of afluorescent dye. There are four fluorescent dyes: blue (B); green (G);yellow (Y); and red (R). Combining dyes together in a reporter complexresults in ten possible two-color combinations (BB, BG, BR, BY, GG, GR,GY, RR, YR, YY). To prevent color-swapping or cross hybridizationbetween different fluorescent dyes, each secondary and tertiary nucleicacid that correspond to a specific fluorescent dye contains a uniquesequence. For example, each tertiary nucleic acid labeled with the Alexa488 fluorophore, or blue color, comprises a complementary sequences onlyto the blue secondary nucleic acid. The blue secondary nucleic acidfurther has a distinct sequence that is complementary only to theprimary nucleic acid molecules that correspond to a color combinationthat includes blue.

Each complementary nucleic acid contains a sequence that is distinctbetween positions R₁, R₂, and R₃ of the barcode domain of the sequencingprobe. Thus, even if positions R₁ and R₂ of the same barcode domainencode for the same dinucleotide, the binding of the complementarynucleic acid molecule that identifies that dinucleotide at position R₁will not bind to position R₂. Likewise, the complementary nucleic acidmolecule that identifies that dinucleotide at position R₂ will not bindto position R₁. Complementary nucleic acids are designed such that theycan be unbound from the sequencing probe efficiently using competitivetoe-hold exchange (for complementary nucleic acids 12 nucleotides inlength) or UV cleavage (for complementary nucleic acids 14 nucleotidesin length).

Preparation of the reporter complex occurs in two sequentialhybridization steps: (1) tertiary nucleic acids to secondary nucleicacids and then (2) tertiary nucleic acids+secondary nucleic acids to theprimary nucleic acid. Four separate tertiary nucleic acid-to-secondarynucleic acid reactions are prepared by combining 100 uM of secondarynucleic acids and 600 uM of tertiary nucleic acids in 4.2×SSPE buffer atroom temperature for 30 minutes. Twenty-four reporter probes are thenprepared separately using 2 uM of primary nucleic acid, 7.2 uM ofsecondary nucleic acid+Dye #1 tertiary nucleic acid, and 7.2 uMsecondary nucleic acid+Dye #2 tertiary nucleic acid in 4.8×SSPE. Thesereactions are heated at 45° C. for 5 minutes and then cooled at roomtemperature for 30 minutes. The 24 reactions are then pooled into threedifferent pools corresponding to the barcode domain (i.e. R₁, R₂, andR₃). For example, eight different reporter probes (2 uM each) binding tothe R₁ barcode domain are pooled together, diluting ten-fold to a finalworking concentration of 200 nM each reporter complex. The reportercomplex can be purified using high pressure liquid chromatography(HPLC). FIG. 39 shows that HPLC purification can remove free oligomersand malformed probes to yield reporter probes.

Following reporter complex preparation is standard testing for qualityassurance. Each of the three pools of reporter probes are tested forbinding to its corresponding barcode region (R₁, R₂, or R₃) in threeseparate flow cells. Testing is performed on a modified sequencing probeconstruct, with only the barcode domain present and immobilized on theflow cell. All eight 12-mers representing each color is multiplexed andall eight reporter probes are expected to be identified with high colorcounts.

To improve the efficiency and accuracy of hybridization of the reporterprobes and the barcode domains of the sequencing probes, various bufferadditives were tested. FIG. 40 shows results from experiments thatindicate buffers containing 5% Dextran Sulfate (500K) and either 15%Formamide or 15% Ethylene Carbonate allow for the most efficient andaccurate hybridization of reporter probes and sequencing probes at shorthybridization times. However, FIG. 41 shows results from experimentsthat indicate Ethylene Carbonate has a negative impact on the surface ofthe sequencing slide, resulting in high loss of target nucleic acidsover time. Thus, buffers containing 5% Dextran Sulfate (500K) and 15%Formamide are superior for efficient and accurate hybridization of thereporter probes and sequencing probes.

Example 11—Design and Validation of Complementary Nucleic Acid Sequences

Reporter probes contain a complementary nucleic acid that binds to aspecific position (R₁, R₂, or R₃) on the barcode domain of a sequencingprobe. Complementary nucleic acids containing either 12 nucleotides(12mer) or 14 nucleotides (14mer) were designed and tested to determineoptimal sequences for hybridization. For screening, the followingcriteria was used to determine optimal sequences: sequences had todisplay high binding efficiency as defined by reporter and sequencingprobe binding at >80% efficiency in 10 sequencing cycles; sequences hadto display fast hybridization kinetics occurring within 15 second to 30seconds; and sequences had to display high specificity with <5%cross-hybridization error in the reporter pool.

Table 4 shows the twenty-four 12mer sequences (SEQ ID NOs: 19-42) thatwere identified. Since each barcode domain contains three positions, thetwenty-four 12mer sequences can be divided into three groups to createan 8×8×8 12mer reporter set.

TABLE 4 Reporter Position 12-mer Sequence Reporter Name Color SEQ ID NO1 AGGACAGATGAC RBB-07 BB 19 1 GTATCGGATGAC R1BG-07d (R1RR-06) BG 20 1AGGAGTGATGAC R1BR-07 BR 21 1 AGGGGTGAGGAG R1GG-07c(R1YR-07) GG 22 1AGAGGGGATGAC R1GR-07 GR 23 1 AGTGGGGAGGAG R1GY-07c(R1BY-07) GY 24 1AGCCGAGATGAC R1RR-07 RR 25 1 AGGGTGGATGAC R1YY-07 YY 26 2 TGGATGGAAAAGR2 BB (forGRv5) BB 27 2 GAAGGAGAAAAG R2 BG (forGYv5) BG 28 2GGGGATGAAAAG R2 BR (forGRv4) BR 29 2 GTGAGGGAAAAG R2 BY (forYYv5) BY 302 AGCCGAGAAAAG R2 GG GG 31 2 CGAGAGGAAAAG R2 GY (forGGv5) GY 32 2GAGGGCGAAAAG R2 RR (forGGv4) RR 33 2 AGCGTGGAAAAG R2 YY YY 34 3TGAGAAGGGTAG RPTR12-BG_Screen3_D2 BG 35 3 GTTGTTATTGTG RPTR12-BR_RC_D4BR 36 3 TTTGGGTTTAGG RPTR12-BY_RC_D3 BY 37 3 GTTAGTGGGAAARPTR12-GR_RC_D7 GR 38 3 ATGGGAAAAAGT RPTR12-GY_RC_D6 GY 39 3GAGTTGGATGAG RPTR12-RR_RC_D10 RR 40 3 ATGTTGTGGGTA RPTR12-YR_RC_D9 YR 413 GAGGGTTTTAAG RPTR12-YY_RC_D8 YY 42

The 14mer sequences were designed in a similar manner but differ fromthe 12mer sequences in three ways. First, 14mer sequences contain alonger hybridization sequence given that 14mer sequences contain 14single stranded nucleotides that bind to a specific position on abarcode domain rather than the 12 single stranded nucleotides present ina 12mer. Second, 14mer sequences contain more sequence diversity becausethey were not designed to accommodate toe-holding-mediated removal.Since 14mer sequences hybridize more strongly to sequencing probes, theefficiency of toe-holding-mediated removal is decreased. Thus, sequenceindependent removal strategies were explored for the 14mer sequences,alleviating sequence constraints during screening. Sequences forscreening were designed using an algorithm that includes the followingset of rules: Nucleotide composition lacking either “G” or “C” (i.e. lowcomplexity sequences); GC content between 40% to 60%; Meltingtemperature (Tm) between 35° C. and 37° C.; Hairpin folding energy(dG)>2; and Compatibility with other sequencing probes (hammingdistance >=7). To minimize the hybridization of 14mer sequences togenomic sequences that can be present in target nucleic acids, potentialsequences were filtered using the External RNA Controls Consortiumsequences as a guide. Third, 14mer sequences were designed to be removedfrom the barcode domains of sequencing probes by strand cleavage usingcleavable linker modifications at the point where the 14mercomplementary nucleic acid is attached to the primary nucleic acid ofthe reporter complex. The removal of the 14mer sequences results in the“darkening” of the reporter complex signal, allowing for the next cycleof sequencing and signal detection to occur. Various cleavable linkermodifications were tested including UV-light cleavable linkers, reducingagent (such as TCEP) cleavable linkers and enzymatically cleavablelinkers (such as uracil cleaved by the USER™ enzyme). All of thesecleavable linker modifications were found to promote efficient reportercomplex darkening. Darkening was further enhanced by the introduction ofcleavable linker modifications into the secondary nucleic acids. Thesecleavable linker modifications were placed between the sequence thathybridizes to the primary nucleic acid and the sequence that hybridizesto the tertiary nucleic acids. FIG. 10 shows the possible positions forcleavable linker modifications within a reporter probe.

Screening of potential 14mer sequences resulted in the identification oftwo groups of acceptable sequences. Table 5 shows the first group, whichcontained 24 sequences (SEQ ID NOs: 43-66). These 24 sequences could besplit into three groups to create an 8×8×8 14mer reporter set.

TABLE 5 Reporter 14-mer Sequence Reporter Name Color SEQ ID AATCTTTTCCCCACT R14-BG_RC-Sc3_B2 BG 43 A CCCCACTATTTCTTRPTR14-BY_Screen4_I2 BY 44 A CTACCCACAACATA RPTR14-YR_Screen3_D9 YR 45 ACCATATAAACCCCA R14-GG_RC-Sc3_B5 GG 46 A AAACTCCAATCTCC R14-GR_RC-Sc3_B7GR 47 A CTATTCTCAACCTA RPTR14-YY_RS0255_H8 YY 48 A CCCCCTCTTTTAAAR14-BB_RC-Sc3_B1 BB 49 A CCAATCTTACCTCA RPTR14-RR_Screen3_B10 RR 50 BCCCTCACATAACTT RPTR14-BG_Screen4_I1 BG 51 B CTCCTCTACTTTCCRPTR14-BB_ERCC_00014.1_10 BB 52 B CCCTAAACCCAAAA RPTR14-BY_Screen3_D3 BY53 B CACTTTTTCCCATC RPTR14-GY_Screen3_D6 GY 54 B CATCTGATTCCTCCR14-RR_ERCC_00042.1_150RC RR 55 B CTAAACCCCCTACT R14-BR_RC-Sc3_B4 BR 56B CCTTTACAAACACA RPTR14-GR_RS0247_H7 GR 57 B ATACCACCCTCTTTRPTR14-YY_Screen3_B8 YY 58 C TATTCTTCTACCCC RPTR14-YR_Screen4_I5 YR 59 CTCTACCCTTCTCAT R14-BG_RC-Sc3_D2 BG 60 C CCACAATAACAACCRPTR14-B4_Screen3_D4 BR 61 C ACCTTAACATTCCC R14-GG_RC-Sc3_D5 GG 62 CATTTCCCACTAACC RPTR14-GR_Screen3_D7 GR 63 C ACTTAAAACCCTCCRPTR14-YY_Screen3_D8 YY 64 C TACCTATTCCTCCA RPTR14-BB_Screen3_D1 BB 65 CCCCCTTTCTCTAAG RPTR14-RR_ERCC_00051.1_220 RR 66

Table 6 shows the other group, which contained 30 sequences (SEQ ID NOs:67-96). These 30 sequences could be split into three groups to create a10×10×10 14mer reporter set.

TABLE 6 SEQ Reporter 14-mer Sequence Reporter Name Color ID AGATGATGGTAGGTG R14_PC_J2_BB_v2 BB 67 A ATGAGAAGGGTAGA R14_PC_D2_BG_v2 BG68 A GTTTTGTTGGTGAG R14_PC_K2_BY_v2 BY 69 A TTAGTGTGTTGGAGR14_PC_K5_BR_v2 BR 70 A ATGTAGGAGAGAGA R14_PC_L1_GG_v2 GG 71 AGGGAATGTTAAGGT R14_PC_D5_GY_v2 GY 72 A GGTTAGTGGGAAAT R14_PC_rcD7_GR_v2GR 73 A GGAGGGTTTTAAGT R14_PC_rcD8_YY_v2 YY 74 A GTAGTGTGGATGTTR14_PC_J5_YR_v2 YR 75 A CTTAGAGAAAGGGG R14_PC_ERCC51_RR_v2 RR 76 BGGAAGAGGATGAAA R14_PC_K1_BB_v2 BB 77 B AAGTTATGTGAGGG R14_PC_spB_BG_v1BG 78 B GGAAAGTAGAGGAG R14_PC_spB_BY_v1 BY 79 B TTTTGGGTTTAGGGR14_PC_spB_BR_v1 BR 80 B AGATGTATGGGTGA R14_PC_L2_GG_v2 GG 81 BGATGGGAAAAAGTG R14_PC_spB_GY_v1 GY 82 B GGAGGAATCAGATG R14_PC_spB_GR_v1GR 83 B AGAGGGATTGATGA R14_PC_J4_YY_v2 YY 84 B TGTGTTTGTAAAGGR14_PC_spB_YR_v1 YR 85 B AAGGAGTGATAGGA R14_PC_J1_RR_v2 RR 86 CTGGTGATTTAGAGG R14_J3_BB_v2 BB 87 C GGGGTAGAAGAATA R14_rcI5_BG_v2 BG 88C AAGAAATAGTGGGG R14_PC_spA_BY_v1 BY 89 C TATGTTGTGGGTAGR14_PC_spA_BR_v1 BR 90 C GTTAAAGGGAGGTT R14_K3_GG_v2 GG 91 CTGGGGTTTATATGG R14_PC_spA_GY_v1 GY 92 C AGGGAATATGGAGA R14_K6_GR_v2 GR93 C TAGGTTGAGAATAG R14_PC_spA_YY_v1 YY 94 C TTTAAAAGAGGGGGR14_PC_spA_YR_v1 YR 95 C TGAGGTAAGATTGG R14_PC_spA_RR_v1 RR 96

After screening, the 8×8×8 12mer, 8×8×8 14mer, and 10×10×10 14merreporter sets were validated experimentally. For the 8×8×8 12mer bindingscheme, validation was performed using a Hyb & Seq prototype to record10 sequencing cycles. Three pools of reporter probes were used in bothlong and short workflow methods. All 512 possible sequencing probebarcode domains were tested. Table 7 shows the experimental steps of thelong and short workflow methods.

TABLE 7 Short workflow: Steps Long workflow: Reporter hyb in One CycleReporter hyb without toehold with toehold 1 Reporter 1 for 15 s, 30 s,or 60 s Reporter 1 for 30 s 2 Image Image 3 Toehold 1 for 60 s to darkReporter 2 + Toehold 1 for 15 s 4 Image Image 5 Reporter 2 for 15 s, 30s, or 60 s Reporter 3 + Toehold 2 for 15 s 6 Image Image 7 Toehold 2 for60 s to dark Wash 8 Image Image 9 Reporter 3 for 15 s, 30 s, or 60 s 10Image 11 Wash 12 Image

Long workflow experiments resulted in >97% darkening efficiency. Forshort workflow experiments, it was assumed that darkening was about asefficient, however it was expected that a small frequency ofnon-darkened reporters would carry over in each image and be miscalledas a new reporter. Indeed, the highest barcode count in the shortworkflow experiment was YYYYYY, which was likely an artifact ofnon-darkening and background. FIG. 42 shows that performance of the8×8×8 12mer reporter set was generally lower in the short workflowcompared to the long workflow. Reporter complex one (which binds toposition R₁ of the barcode domain) and reporter complex three (whichbinds to position R₃ of the barcode domain) had lower efficiencies inthe short workflow compared to long workflow. This is expected forreporter complex three since it includes eight additional toe-holdoligonucleotides, at a high concentrations of 2.5 uM each, which caninterfere with reporter hybridizations. Reporter complex one shouldbehave similarly between the two workflows, as no toe-holds were used toremove the first reporter complex in either the short or long workflows.Total error was also higher (1.3- to 2-fold) in the short workflowcompared to long workflow for all three reporter probes.

The 8×8×8 14mer reporter set was validated by testing the efficiency,specificity, and speed of hybridization to all 512 possible sequencingprobe barcode domains. The sequencing probe barcode domains wereimmobilized directly onto the glass of a Hyb & Seq sequencing cartridge.FIG. 43 shows that the 8×8×8 14mer reporter probes hybridized with anaverage efficiency of 88% in only 15 seconds with an average error rateof 5.1%. The majority of this error is due to incorrect identificationof the reporter not due to incorrect hybridization. Misclassificationerror of reporters remains the largest component of reporter error.

The 10×10×10 14mer reporter set was validated by testing for efficiency,specificity, and speed of hybridization to 30 complementary, truncatedsequencing probe barcode domains. Each barcode domain contained only onereporter binding site. These barcode domains were immobilized directlyonto the glass of a Hyb & Seq sequencing cartridge. FIG. 44 shows thatthe 10×10×10 14mer reporter set hybridized with an average efficiency of90% in only 15 seconds with an average error rate of 5.0%. Again, thevast majority of error was due to incorrect identification of thereporter not due to incorrect hybridization.

Example 12—Design and Testing of Standard and Three-Part SequencingProbes

The target binding and barcode domains of a sequencing probe areseparated by a double-stranded “stem”. FIG. 2 shows two sequencing probearchitectures that were experimentally tested. On a standard sequencingprobe, the target binding and barcode domains are present on the sameoligonucleotide, which binds to a stem oligonucleotide to create a 36nucleotide long double-stranded region. Using this architecture, eachsequencing probes in a pool of probes use the same stem sequence. On athree-part probe, the target binding and barcode domains are separateDNA oligonucleotides that are bound together by a 36 nucleotide stemoligonucleotide. To prevent possible exchange of barcode domains, eachbarcode has a unique stem sequence and are hybridized separately beforepooling sequencing probes.

FIG. 45 shows the results of a series of experiment performed to comparethree-part sequencing probes to standard sequencing probes. Theseexperiments confirmed that three-part sequencing probes survive anentire sequencing cycle with ˜80% of all reads for both configurationsincluding the detection of the third reporter probe. When compared tostandard sequencing probes, three-part probes show ˜12% fewer counts. Tostudy the propensity for exchange of the barcode domain oligo, a highconcentration of a short alternative oligonucleotide containing the samestem sequence was added to the reaction. The results indicated that ˜13%of detected three-part sequencing probes had exchanged barcode oligoes.Oligonucleotide exchange will need to be mitigated with theincorporation of unique stem sequences. Despite the slight reduction inperformance, three-part probes provide the benefits of designflexibility, speedy oligo synthesis, and reduced cost.

Example 13—Effect of Locked Nucleic Acid Substitutions in the TargetBinding Domain

The effect of the substitution of locked nucleic acids (LNAs) into thetarget binding domain of sequencing probes was tested as follows.Sequencing probes were hybridized to reporter probes in solution andproperly formed sequencing probe-reporter probes were purified. Thesequencing probe-reporter probes were then hybridized to synthetictarget nucleic acids in solution and loaded onto a prototype sequencingcartridge. The synthetic target nucleic acid consisted of 50 nucleotidesand was biotinylated. Sequencing probes were tested either individuallyor in a pool of nine. For the pool of nine sequencing probes, the probeswere designed to bind along the length of the target nucleic acid. Foranalysis, the entire reaction was deposited by a breadboard instrumentonto a streptavidin-coated cover slide and then flow stretched. Thereporter probes were then imaged and counted using the appropriateinstrument and software, for example with the NanoString nCounter®instrument and software.

Each sequencing probe contained a target binding domain of 10nucleotides (SEQ ID NO: 97). LNA substitutions within the target bindingdomains were made to include 2, 3, or 4 LNA bases at the positions shownin FIG. 46. FIG. 46 shows that the binding affinity of the individualsequencing probes for the target nucleic acid increased as the number ofLNA bases increased. Importantly, FIG. 46 shows that the incorporationof LNA bases did not decrease the specificity of sequence probe binding.The pool of nine sequencing probes was tested to determine base coveragewhen probes could compete for target binding. FIG. 47 shows that when asingle LNA probe was introduced into the pool, the coverage of theaffected bases was increased with little effect on the binding ofsurrounding probes. These results indicated that LNA base substitutionscan improve base sensitivity without reducing specificity.

Example 14—Effect of Modified Nucleotide and Nucleic Acid AnalogueSubstitutions in the Target Binding Domain

The effect of the substitution of various modified nucleotides andnucleic acid analogues, including locked nucleic acids (LNA), bridgednucleic acids (BNA), propyne-modified nucleic acids, zip nucleic acids(ZNA®), isoguanine and isocytosine, into the target binding domain ofsequencing probes was tested as follows. Biotinylated target nucleicacids 50 nucleotides in length were loaded onto a streptavidin coverslide of a prototype sequencing cartridge. Sequencing and reporterprobes were then sequentially introduced into the sample chamber andimaged using a Hyb & Seq prototype instrument. The images were processedto compare the counts for each different sequencing probe. Substitutionsin the 10 nucleotide (SEQ ID NO: 99) target binding domain of thesequencing probes were made to include LNA, BNA, propyne, and ZNA® basesat the positions shown in FIG. 48. FIG. 48 shows that probes containingLNAs and BNAs showed the largest increase in binding affinity whilemaintaining specificity, as indicated by the number of counts detectedfor matching and mismatched targets. These results indicated that LNA orBNA base substitutions can improve base sensitivity without reducingspecificity.

Example 15—Determining Accuracy of the Sequencing Method of the PresentDisclosure

FIG. 49 depicts the results from an experiment that quantified the rawspecificity of the sequencing method of the present disclosure. In thisexperiment, a sequencing reaction was performed in which a pool of 4different sequencing probes was hybridized to a target nucleic acid thatincluded a fragment of NRAS exon2 (SEQ ID NO: 1). Each sequencing probe(barcode 1 to 4) had a target binding domain that was identical exceptthat the hexamer of the target binding domain differed at position b₅,as depicted in the top panel of FIG. 49. In this example, barcode 4 isthe correct sequencing probe. After hybridization of the sequencingprobes, reporter probes were sequentially hybridized to each of thethree positions of the barcode domain (R₁, R₂ and R₃) and thecorresponding fluorescence data recorded. The middle panel of FIG. 49depicts the number of times each color combination was recorded for thethree barcode domain positions and the percentage of the time that thecorrect combination was recorded. The color combination at R₁ wascorrectly identified 96% of the time, the color combination at R₂ wascorrectly identified 97% of the time and the correct color combinationat R₃ was correctly identified 94% of the time. As depicted in thebottom panel of FIG. 49, this leads to an overall raw specificity of94%. The sources of error that could explain the miscalled barcodedomain positions include: (a) non-specific binding of reporter probes tothe surface of the flow cell and (b) incorrect hybridization of reporterprobes. The estimated amount of reporter hybridization errors wasapproximately 2 to 4%.

FIG. 50 shows the results from an experiment to determine the accuracyof the sequencing method of the present disclosure when nucleotides inthe target nucleic acid are sequenced by more than one sequencing probe.As shown in the top panel of FIG. 50, the target nucleic acid in thisexample is a fragment of NRAS exon2 (SEQ ID NO: 1). The particular baseof interest is a cytosine (C) that is highlighted in the target nucleicacid. The base of interest will be hybridized to two differentsequencing probes, each with a distinct footprint of hybridization tothe target nucleic acid. In this example, sequencing probes 1 to 4(barcode 1 to 4) bind three nucleotides to the left of the base ofinterest, while sequencing probes 5 to 8 (barcodes 5 to 8) bind 5nucleotides to the left of the base of interest. The middle panel ofFIG. 50 shows the number of times specific color combinations wererecorded at each position of the barcode domains of the sequencingprobes. After image quantification and using the base calling techniquesdepicted in FIG. 26, an average accuracy of 98.98% can be recorded.

What is claimed is:
 1. A complex comprising a) a composition comprisinga target binding domain and a barcode domain, wherein the target bindingdomain comprises at least eight nucleotides and is capable of binding atarget nucleic acid, wherein at least six nucleotides in the targetbinding domain are capable of identifying a corresponding nucleotide inthe target nucleic acid molecule and wherein at least two nucleotides inthe target binding domain do not identify a corresponding nucleotide inthe target nucleic acid molecule; wherein at least two nucleotides ofthe at least six nucleotides in the target binding domain are modifiednucleotides or nucleotide analogues; wherein the barcode domaincomprises a synthetic backbone, the barcode domain comprising at leastthree attachment positions, each attachment position comprising at leastone attachment region comprising at least one nucleic acid sequencecapable of being bound by a complementary nucleic acid molecule, whereinthe nucleic acid sequence of the at least three attachment positionsdetermines the position and identity of the at least six nucleotides inthe target nucleic acid that is bound by the target binding domain, andwherein each of the at least three attachment positions have a differentnucleic acid sequence; and a first complementary primary nucleic acidmolecule hybridized to a first attachment position of the at least threeattachment positions, wherein the first primary complementary nucleicacid molecule comprises at least two domains and a linker modification,wherein the first domain is hybridized to the first attachment positionof the barcode domain and the second domain capable of hybridizing to atleast one complementary secondary nucleic acid molecule, and wherein thelinker modification is

and wherein the linker modification is located between the first andsecond domains.
 2. A complex comprising a) a composition comprising atarget binding domain and a barcode domain, wherein the target bindingdomain comprises at least eight nucleotides and is capable of binding atarget nucleic acid, wherein at least six nucleotides in the targetbinding domain are capable of identifying a corresponding nucleotide inthe target nucleic acid molecule and wherein at least two nucleotides inthe target binding domain do not identify a corresponding nucleotide inthe target nucleic acid molecule; wherein at least two nucleotides ofthe at least six nucleotides in the target binding domain are modifiednucleotides or nucleotide analogues; wherein the barcode domaincomprises a synthetic backbone, the barcode domain comprising at leastthree attachment positions, each attachment position comprising at leastone attachment region comprising at least one nucleic acid sequencecapable of being bound by a complementary nucleic acid molecule, whereinthe nucleic acid sequence of the at least three attachment positionsdetermines the position and identity of the at least six nucleotides inthe target nucleic acid that is bound by the target binding domain,wherein each attachment position of the at least three attachmentpositions corresponds to two nucleotides of the at least six nucleotidesin the target binding domain and each of the at least three attachmentpositions have a different nucleic acid sequence, and wherein thenucleic acid sequence of each position of the at least three attachmentpositions determines the position and identity of the corresponding twonucleotides of the at least six nucleotides in the target nucleic acidthat is bound by the target binding domain; and a first complementaryprimary nucleic acid molecule hybridized to a first attachment positionof the at least three attachment positions, wherein the first primarycomplementary nucleic acid molecule comprises at least two domains and alinker modification, wherein the first domain is hybridized to the firstattachment position of the barcode domain and the second domain capableof hybridizing to at least one complementary secondary nucleic acidmolecule, and wherein the linker modification is

and wherein the linker modification is located between the first andsecond domains.
 3. A probe comprising a target binding domain and abarcode domain; wherein the target binding domain comprises at leasteight nucleotides and is capable of binding a target nucleic acid,wherein at least six nucleotides in the target binding domain arecapable of identifying a corresponding nucleotide in the target nucleicacid molecule and wherein at least two nucleotides in the target bindingdomain do not identify a corresponding nucleotide in the target nucleicacid molecule; wherein at least two nucleotides of the at least sixnucleotides in the target binding domain are modified nucleotides ornucleotide analogues; wherein the barcode domain comprises a syntheticbackbone, the barcode domain comprising at least three attachmentpositions, each attachment position comprising at least one attachmentregion comprising at least one nucleic acid sequence capable of beingbound by a complementary nucleic acid molecule, wherein the nucleic acidsequence of the at least three attachment positions determines theposition and identity of the at least six nucleotides in the targetnucleic acid that is bound by the target binding domain, and whereineach of the at least three attachment positions have a different nucleicacid sequence.
 4. A probe comprising a target binding domain and abarcode domain; wherein the target binding domain comprises at leasteight nucleotides and is capable of binding a target nucleic acid,wherein at least six nucleotides in the target binding domain arecapable of identifying a corresponding nucleotide in the target nucleicacid molecule and wherein at least two nucleotides in the target bindingdomain do not identify a corresponding nucleotide in the target nucleicacid molecule; wherein at least two nucleotides of the at least sixnucleotides in the target binding domain are modified nucleotides ornucleotide analogues; wherein the barcode domain comprises a syntheticbackbone, the barcode domain comprising at least three attachmentpositions, each attachment position comprising at least one attachmentregion comprising at least one nucleic acid sequence capable of beingbound by a complementary nucleic acid molecule, wherein each attachmentposition of the at least three attachment positions corresponds to twonucleotides of the at least six nucleotides in the target binding domainand each of the at least three attachment positions have a differentnucleic acid sequence, and wherein the nucleic acid sequence of eachposition of the at least three attachment positions determines theposition and identity of the corresponding two nucleotides of the atleast six nucleotides in the target nucleic acid that is bound by thetarget binding domain.
 5. The probe of claim 3, wherein said syntheticbackbone comprises a polysaccharide, a polynucleotide, a peptide, apeptide nucleic acid, or a polypeptide.
 6. The probe of claim 3, whereinsaid synthetic backbone comprises a single-stranded DNA.
 7. The probe ofclaim 3, wherein said sequencing probe comprises a single-stranded DNAsynthetic backbone and a double-stranded DNA spacer between the targetbinding domain and the barcode domain.
 8. The sequencing probe of claim7, wherein said double-stranded DNA spacer is from about 1 nucleotide toabout 100 nucleotides in length.
 9. The sequencing probe of claim 7,wherein said double-stranded DNA spacer is about 36 nucleotides inlength.
 10. The probe of claim 3, wherein the number of nucleotides inthe target binding domain is greater than the number of attachmentpositions in the barcode domain.
 11. The probe of claim 3, wherein thenumber of nucleotides in the target binding domain is at least five morethan the number of attachment positions in the barcode domain.
 12. Theprobe of claim 3, wherein the target binding domain comprises eightnucleotides and the barcode domain comprises three attachment positions.13. The probe of claim 3, wherein at least four nucleotides of the atleast six nucleotides in the target binding domain are modifiednucleotides or nucleotide analogues.
 14. The probe of claim 3, whereinat least six nucleotides of the at least six nucleotides in the targetbinding domain are modified nucleotides or nucleotide analogues.
 15. Theprobe of claim 3, wherein the modified nucleotide or nucleic acidanalogue is a locked nucleic acid (LNA).
 16. The probe of claim 3,wherein at least one of the at least two nucleotides in the targetbinding domain that do not identify a corresponding nucleotide in thetarget nucleic acid molecule precedes the at least six nucleotides inthe target binding domain and wherein at least one of the at least twonucleotides in the target binding domain do not identify a correspondingnucleotide in the target nucleic acid molecule follows the at least sixnucleotides in the target binding domain.
 17. The probe of claim 3,wherein each attachment position in the barcode domain comprises thesame number of attachment regions.
 18. The probe of claim 3, wherein atleast one of the at least three attachment positions in the barcodedomain comprises a different number of attachment regions than the othertwo.
 19. The probe of claim 3, wherein each attachment position in thebarcode domain comprises one attachment region.
 20. The probe of claim3, wherein each attachment position in the barcode domain comprises morethan one attachment region.
 21. The probe of claim 3, wherein when theattachment position in the barcode domain comprises more than oneattachment region, the attachment regions are the same.
 22. The probe ofclaim 3, wherein when the attachment position in the barcode domaincomprises more than one attachment region, the attachment regions aredifferent.
 23. The probe of claim 3, wherein each nucleic acid sequencecomprising each attachment region in the barcode domain is from about 8nucleotides to about 20 nucleotides in length.
 24. The probe of claim 3,wherein each nucleic acid sequence comprising each attachment region inthe barcode domain is about 12 nucleotides in length.
 25. The probe ofclaim 3, wherein each nucleic acid sequence comprising each attachmentregion in the barcode domain is about 14 nucleotides in length.
 26. Theprobe of claim 3, wherein at least one of the at least three attachmentpositions in the barcode domain is adjacent to at least one flankingsingle-stranded polynucleotide.
 27. The probe of claim 3, wherein eachof the at least three attachment positions in the barcode domain isadjacent to at least one flanking single stranded polynucleotide. 28.The probe of claim 3, wherein at least one attachment region in at leastone attachment position is integral to the synthetic backbone.
 29. Theprobe of claim 3, wherein each attachment region in each of the at leastthree attachment positions is integral to the synthetic backbone. 30.The probe of claim 3, wherein the complementary nucleic acid molecule isRNA, DNA or PNA.
 31. The probe of claim 30, wherein the complementarynucleic acid molecule is DNA.
 32. The probe of claim 3, wherein thecomplementary nucleic acid molecule is a primary nucleic acid molecule,wherein the primary nucleic acid molecule directly binds to at least oneattachment region within at least one attachment position of the barcodedomain.
 33. The sequencing probe of claim 32, wherein the primarynucleic acid molecule comprises at least two domains, a first domaincapable of binding to at least one attachment region within at least oneattachment position of the barcode domain and a second domain capable ofbinding to at least one complementary secondary nucleic acid molecule.34. The probe of claim 33, wherein the primary nucleic acid moleculecomprises a cleavable linker.
 35. The probe of claim 34, wherein thecleavable linker is located between the first domain and the seconddomain.
 36. The probe of claim 34, wherein the linker isphoto-cleavable.
 37. The probe of claim 32, wherein the primary nucleicmolecule is hybridized to at least one attachment region within at leastone attachment position of the barcode domain and comprises a firstdetectable label and an at least second detectable label.
 38. The probeof claim 33, wherein the primary nucleic molecule is hybridized to atleast one attachment region within at least one attachment position ofthe barcode domain and is hybridized to at least one secondary nucleicacid molecule.
 39. The probe of claim 38, wherein the primary nucleicmolecule is hybridized to at least one, two, three, four, five, or moresecondary nucleic acid molecules.
 40. The probe of claim 38, wherein theprimary nucleic molecule is hybridized to four secondary nucleic acidmolecules.
 41. The probe of claim 37, wherein the first and at leastsecond detectable labels have the same emission spectrum or havedifferent emission spectra.
 42. The probe of claim 33, wherein thesecondary nucleic acid molecule comprises at least two domains, a firstdomain capable of binding to complementary sequence in at least oneprimary nucleic acid molecule; and a second domain capable of binding to(a) a first detectable label and an at least second detectable label,(b) to at least one complementary tertiary nucleic acid molecule, or (c)a combination thereof.
 43. The probe of claim 42, wherein the secondarynucleic acid molecule comprises a cleavable linker.
 44. The probe ofclaim 43, wherein the cleavable linker is located between the firstdomain and the second domain.
 45. The probe of claim 43, wherein thelinker is photo-cleavable.
 46. The probe of claim 42, wherein thesecondary nucleic molecule is hybridized to at least one primary nucleicacid molecule; and comprises a first detectable label and an at leastsecond detectable label.
 47. The probe of claim 42, wherein thesecondary nucleic molecule is hybridized to at least one primary nucleicacid molecule and is hybridized to at least one tertiary nucleic acidmolecule.
 48. The probe of claim 42, wherein the secondary nucleicmolecule is hybridized to (a) at least one primary nucleic acidmolecule, (b) at least one tertiary nucleic acid molecule, and (c) afirst detectable label and an at least second detectable label.
 49. Theprobe of claim 47, wherein the secondary nucleic acid molecule ishybridized to at least one, two, three, four, five, six, seven, or moretertiary nucleic acid molecules.
 50. The probe of claim 47, wherein thesecondary nucleic molecule is hybridized to one tertiary nucleic acidmolecule.
 51. The probe of claim 46, wherein the first and at leastsecond detectable labels have the same emission spectrum or havedifferent emission spectra.
 52. The probe of claim 42, wherein thetertiary nucleic acid molecule comprises at least two domains, a firstdomain capable of binding to complementary sequence in a secondarynucleic acid molecule; and a second domain capable of binding to a firstdetectable label and an at least second detectable label.
 53. The probeof claim 52, wherein the tertiary nucleic acid molecule comprises acleavable linker.
 54. The probe of claim 53, wherein the cleavablelinker is located between the first domain and the second domain. 55.The probe of claim 53, wherein the linker is photo-cleavable.
 56. Theprobe of claim 52, wherein the tertiary nucleic molecule is hybridizedto at least one secondary nucleic acid molecule and comprises a firstdetectable label and an at least second detectable label.
 57. The probeof claim 52, wherein the first and at least second detectable labelshave the same emission spectrum or have different emission spectra. 58.The probe of claim 55, wherein the at least first and second detectablelabels located on the secondary nucleic acid molecule have the sameemission spectra and the at least first and second detectable labelslocated on the tertiary nucleic acid molecule have the same emissionspectra, and wherein the emission spectra of the detectable labels onthe secondary nucleic acid molecule are different than the emissionspectra of the detectable labels on the tertiary nucleic acid molecule.59. A population of probes comprising a plurality of the probe of claim3.
 60. A method for determining a nucleotide sequence of a nucleic acidcomprising (1) hybridizing a first probe of claim 3 to a first region ofa target nucleic acid that is optionally immobilized to a substrate atone or more positions; (2) hybridizing a first complementary nucleicacid molecule comprising a first detectable label and a seconddetectable label to a first attachment position of the at least threeattachment positions of the barcode domain; (3) identifying the firstand the second detectable label of the first complementary nucleic acidmolecule hybridized to the first attachment position; (4) removing thefirst and the second detectable label hybridized to the first attachmentposition; (5) hybridizing a second complementary nucleic acid moleculecomprising a third detectable label and a fourth detectable label to asecond attachment position of the at least three attachment positions ofthe barcode domain; (6) identifying the third and the fourth detectablelabel of the second complementary nucleic acid molecule hybridized tothe second attachment position; (7) removing the third and fourthdetectable label hybridized to the second attachment position; (8)hybridizing a third complementary nucleic acid molecule comprising afifth detectable label and a sixth detectable label to a thirdattachment position of the at least three attachment positions of thebarcode domain; (9) identifying the fifth and the sixth detectable labelof the third complementary nucleic acid molecule hybridized to the thirdattachment position; and (10) identifying the linear order of at leastsix nucleotides in the first region of the optionally immobilized targetnucleic acid hybridized to the target binding domain of the sequencingprobe based on the identity of the first detectable label, seconddetectable label, third detectable label, fourth detectable label, fifthdetectable label and sixth detectable label.
 61. The method of claim 60,wherein the target nucleic acid is obtained from a predetermined gene.62. The method of claim 60, wherein steps (4) and (5) occur sequentiallyor concurrently.
 63. The method of claim 60, wherein the first andsecond detectable labels have the same emission spectrum or havedifferent emission spectra.
 64. The method of claim 60, wherein thethird and fourth detectable labels have the same emission spectrum orhave different emission spectra.
 65. The method of claim 60, wherein thefifth and sixth detectable labels have the same emission spectrum orhave different emission spectra.
 66. The method of claim 60, wherein thefirst complementary nucleic acid molecule comprises a cleavable linker.67. The method of claim 60, wherein the second complementary nucleicacid molecule comprises a cleavable linker.
 68. The method of claim 60,wherein the second complementary nucleic acid molecule
 69. The method ofclaim 60, wherein the first complementary nucleic acid molecule, thesecond complementary nucleic acid molecule and the third complementarynucleic acid molecule each comprise a cleavable linker.
 70. The methodof claim 66, wherein the cleavable linker is photo-cleavable.
 71. Themethod of claim 70, wherein removing the first complementary nucleicacid molecule comprises contacting the first complementary nucleic acidmolecule with light.
 72. The method of claim 71, wherein the light isprovided by a light source selected from the group consisting of anarc-lamp, a laser, a focused UV light source, and light emitting diode.73. The method of claim 60, further comprising (11) removing the atleast first sequencing probe from the first region of the optionallyimmobilized target nucleic acid; (12) hybridizing at least a secondprobe of claim 3 to a second region of the target nucleic acid that isoptionally immobilized to a substrate at one or more positions, andwherein the target binding domain of the first sequencing probe and theat least second sequencing probe are different; (13) hybridizing a firstcomplementary nucleic acid molecule comprising a first detectable labeland a second detectable label to a first attachment position of the atleast three attachment positions of the barcode domain; (14) detectingthe first and the second detectable label of the first complementarynucleic acid molecule hybridized to the first attachment position; (15)hybridizing a second complementary nucleic acid molecule comprising athird detectable label and a fourth detectable label to a secondattachment position of the at least three attachment positions of thebarcode domain; (16) detecting the third and the fourth detectable labelof the second complementary nucleic acid molecule hybridized to thesecond attachment position; (17) removing the third and fourthdetectable label hybridized to the second attachment position; (18)hybridizing a third complementary nucleic acid molecule comprising afifth detectable label and a sixth detectable label to a thirdattachment position of the at least three attachment positions of thebarcode domain; (19) identifying the fifth and the sixth detectablelabel of the third complementary nucleic acid molecule hybridized to thethird attachment position; and (20) identifying the linear order of atleast six nucleotides in the second region of the optionally immobilizedtarget nucleic acid hybridized to the target binding domain of the atleast second sequencing probe based on the identity of the firstdetectable label, second detectable label, third detectable label,fourth detectable label, fifth detectable label and sixth detectablelabel.
 74. The method of claim 73, further comprising assembling eachidentified linear order of nucleotides in the at least first region andat least second region of the optionally immobilized target nucleicacid, thereby identifying a sequence for the optionally immobilizedtarget nucleic acid.
 75. An apparatus for performing the method of claim60.
 76. A kit comprising a substrate, a population of probes of claim59, at least one capture probe, at least three complementary nucleicacid molecules comprising a first detectable label and at least twosecond detectable labels, and instructions for use.